CN113209136A

CN113209136A - Composition of injectable hydrogel and genetically engineered cells and application thereof in treating OA

Info

Publication number: CN113209136A
Application number: CN202110484793.9A
Authority: CN
Inventors: 章雪晴; 于伟
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-08-06
Anticipated expiration: 2041-04-30
Also published as: CN113209136B

Abstract

The present invention provides the use of an injectable hydrogel and a composition of genetically engineered cells for the treatment of osteoarthritis. Specifically, the composition comprises: (a) genetically engineered modified ADSCs; and (b) an injectable hydrogel carrier encapsulating the genetically engineered ADSCs, wherein the genetically engineered ADSCs are genetically engineered ADSCs overexpressing TGF-beta 1. The composition of the present invention has excellent cartilage and subchondral bone protecting effects and anti-inflammatory effects, and is particularly suitable for the prevention and/or treatment of osteoarthritis.

Description

Composition of injectable hydrogel and genetically engineered cells and application thereof in treating OA

Technical Field

The invention relates to the field of biomedicine, in particular to application of an injectable hydrogel and a composition of genetically engineered cells in treating OA.

Background

Osteoarthritis (OA) is a common chronic degenerative joint disease, about 3 million people all over the world suffer from OA, which is an important cause of disability of the elderly, and joint pain and loss of function caused by OA seriously affect the quality of life of patients. The pathological features of OA are mainly manifested by degenerative changes in the articular cartilage, synovial inflammation, synovial hyperplasia, osteophyte formation and subchondral bone sclerosis. It is generally considered to be an "abrasive" disease characterized by damage to articular cartilage, however inflammation is often a critical factor associated with increased levels of cartilage damage and affects the physiological function of the entire joint. The reason for this is mainly because inflammatory factors such as TNF- α and IL-1 β in the joints affect the normal function of chondrocytes and promote the expression of inflammation-related catabolic genes, thereby damaging the extracellular matrix (ECM) of chondrocytes.

As a potential therapeutic agent, ADSCs (adipamt-derived stem cells, ADSCs) have recently been extensively studied to reduce intra-articular inflammation and slow the progression of articular cartilage damage. They can paracrine large amounts of anti-inflammatory mediators and chondroprotective compounds. There is a need in the art to provide better methods and medicaments for treating OA.

Disclosure of Invention

The object of the present invention is to provide a pharmaceutical composition for preventing and/or treating osteoarthritis having excellent cartilage, subchondral bone protecting effects and anti-inflammatory effects.

In a first aspect of the present invention, there is provided a use of a pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of osteoarthritis, wherein the pharmaceutical composition comprises:

(a) genetically engineered modified adipose stem cells (ADSCs); and

(b) an injectable hydrogel carrier encapsulating the genetically modified adipose-derived stem cells,

wherein, the fat stem cell modified by the genetic engineering is a genetically engineered fat stem cell (T-ADSCs) over-expressing transforming growth factor-beta 1 (TGF-beta 1).

In another preferred embodiment, the osteoarthritis is primary osteoarthritis or secondary osteoarthritis.

In another preferred embodiment, the prevention and/or treatment of osteoarthritis comprises one or more characteristics selected from the group consisting of:

(a) increasing bone volume, or decreasing decrease in bone volume;

(b) increasing the bone volume/tissue volume (BV/TV) ratio;

(c) increasing trabecular number (tb.n), or decreasing the drop in tb.n;

(d) slowing down articular cartilage lesions;

(e) slowing down the bone loss;

(f) increasing bone density;

(g) slowing down the decrease of aggrecan content in cartilage;

(h) slowing down the reduction of the type II collagen content in the cartilage;

(i) slowing the inflammatory response in the joint space; and/or

(j) Reducing TNF-alpha levels in joint synovial fluid.

In another preferred embodiment, the cartilage disorder comprises cartilage damage, chondrocyte senescence, or a combination thereof.

In another preferred embodiment, the genetically engineered adipose stem cells overexpressing TGF- β 1 may overexpress TGF- β 1 in human, rat, and/or mouse, and/or an amino acid sequence having at least 80%, 90%, 95%, or 99% identity to the TGF- β 1 amino acid sequence in human, rat, and/or mouse.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) genetically engineered modified ADSCs; and

(b) an injectable hydrogel carrier encapsulating the genetically engineered ADSCs,

wherein the ADSCs modified by genetic engineering are genetically engineered ADSCs over-expressing TGF-beta 1.

In another preferred embodiment, the hydrogel is composed of one or more materials selected from the group consisting of: polycarboxymethyl cellulose, alginates, hydroxypropylmethyl cellulose, carboxymethyl cellulose, ethyl hydroxyethyl cellulose, hydroxyalkyl cellulose, alkyl cellulose, polylactic acid, microcrystalline cellulose, polylactic-co-glycolic acid, dextrin, hydroxyethyl starch, hydroxyethyl chitosan, Hyaluronic Acid (HA), collagen, gelatin, and derivatives of hyaluronic acid, collagen, gelatin.

In another preferred embodiment, the hydrogel is a cell matrix-like hydrogel based on HA and type I collagen (collagen I, Col I).

In another preferred embodiment, the hydrogel is self-assembled by type I collagen and contains sulfhydryl groups (e.g., -CH)₂CH₂SH) from HA (HA-SH).

In another preferred embodiment, the amount of HA-SH in the hydrogel is 1 to 5 wt%, such as 2 wt%, 3 wt%, 3.5 wt% or 4 wt%, based on the total weight of the hydrogel.

In another preferred embodiment, the concentration of the thiol group of HA-SH is (1-5). times.10^-4mol g^-1Preferably, (2-4). times.10^-4mol g^-1E.g. 3X 10^-4mol g^-1、3.5×10^-4mol g^-1、3.98×10^-4mol g^-1、4×10^-4 mol g^-1Or 4.5X 10^-4mol g^-1。

In another preferred embodiment, the HA HAs a Mw of 50-200kDa, preferably 80-120kDa, such as 80kDa, 100kDa or 110 kDa.

In another preferred embodiment, the collagen type I is present in the hydrogel in an amount of 0.1-2mg/mL, such as 0.8-1.5mg/mL, 1mg/mL, or 1.2mg/mL, based on the total weight of the hydrogel.

In another preferred embodiment, the content of the T-ADSCs in the hydrogel is 0.1-10 × 10⁶cells/mL, preferably 0.5 to 5X 10⁶cells/mL, e.g. 1X 10⁶cell/mL, 2X 10⁶cell/mL, 4X 10⁶cells/mL.

In another preferred example, the expression level of TGF-beta 1 of the T-ADSCs is increased by 20% or more, 30% or more, 40% or more, 50% or more, 80% or more, or 100% or more in terms of cumulative expression amount, compared with the ADSCs before genetic engineering under the same experimental conditions.

In another preferred embodiment, the genetically engineered ADSCs overexpressing TGF- β 1 are obtained by transfecting ADSCs with TGF- β 1 overexpressing plasmid DNA (e.g., pEGFP-TGF- β 1).

In another preferred embodiment, the transfected delivery vehicle is Lipo 2000, Lipo 3000, dendrimer, polyethyleneimine or poly (β -amino ester).

In another preferred embodiment, the delivery vehicle is poly (β -amino ester), PBAE, preferably with a mass ratio of poly (β -amino ester) to plasmid DNA of 10-120:1, preferably 60-90: 1.

In another preferred embodiment, the ADSCs used to prepare the genetically engineered ADSCs overexpressing TGF- β 1 are from: autologous, allogeneic, xenogeneic, or a combination thereof.

In another preferred embodiment, the pharmaceutical composition is prepared by the following method, comprising the steps of:

(i) providing a mixed solution of T-ADSCs, HA-SH and type I collagen; and

(ii) adjusting the pH value of the solution to 7.4-8.0, and reacting to form hydrogel, thereby obtaining the pharmaceutical composition. And the T-ADSCs are dispersed in the solution after the pH is adjusted.

In another preferred embodiment, in step (ii), the hydrogel-forming reaction conditions include one or more of the following characteristics:

the temperature of the reaction is 37 +/-2 ℃, preferably 37 +/-1 ℃; and/or

The reaction time is 1-30min, preferably 2-8min, such as 3, 4, 5, 6 or 7 min.

In another preferred embodiment, the hydrogel is a gel formed in physiological saline, water for injection, or a neutral physiologically isotonic phosphate buffer.

In another preferred embodiment, the pharmaceutical composition further comprises a second therapeutic agent selected from the group consisting of: anti-osteoporosis drugs (such as bisphosphonates (Clodronate, osteochosphine), Pamidronate (Pamidronate) and Tiludronate (Tiludronate)), Alendronate (Alendronate, Formoset, Fosamax) and Risedronate (Risedronate), Ibandronate (Boniva), Zoledronic acid (Reclean)), parathyroid hormone, prostaglandin E2, estrogens, estrogen receptor modulators, strontium salts (such as strontium ranelate), non-steroidal anti-inflammatory drugs (acetaminophen, diclofenac sodium, ibuprofen sustained release capsules, meloxicam), triamcinolone acetonide, tofacitinib, anti-inflammatory analgesics, antibacterial drugs, antiviral drugs, or combinations thereof.

In another preferred embodiment, the pharmaceutical composition is a gel injection.

In a third aspect of the present invention, an application of genetically engineered ADSCs in prevention and/or treatment of osteoarthritis is provided, wherein the genetically engineered ADSCs are genetically engineered ADSCs that overexpress TGF- β 1.

In another preferred embodiment, the T-ADSCs are delivered via an injectable hydrogel carrier.

(a) increasing bone volume, or decreasing decrease in bone volume;

(b) increasing the bone volume/tissue volume (BV/TV) ratio;

(c) increasing the number of trabeculae, or decreasing the decrease in the number of trabeculae;

(d) slowing down articular cartilage lesions;

(e) slowing down the bone loss;

(f) increasing bone density;

(g) slowing down the decrease of aggrecan content in cartilage;

(i) slowing the inflammatory response in the joint space; and/or

(j) Reducing TNF-alpha levels in joint synovial fluid.

In a fourth aspect, the present invention provides a method for preventing and/or treating osteoarthritis, comprising the steps of:

genetically engineered ADSCs overexpressing TGF- β 1 or a pharmaceutical composition comprising the same are administered to a subject in need thereof, thereby preventing and/or treating osteoarthritis.

In another preferred embodiment, the administration is by injection or implantation to the affected joint area.

In another preferred embodiment, the pharmaceutical composition is as described in the second aspect of the invention.

In another preferred embodiment, the subject is a mammal, such as a human, rat, mouse, monkey, cat or dog.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a schematic diagram of one embodiment of the present invention. Injectable ECM-like hydrogels were prepared by HA-SH self-crosslinking and Col I self-assembly. T-ADSCs are encapsulated in the hydrogel and delivered to the knee joint of OA model rats by joint cavity injection or transplantation. The injectable degradable hydrogel provides a suitable 3D microenvironment for cell growth, and the T-ADSCs combine the treatment effects of TGF-beta 1 and ADSCs, so that the purpose of effectively treating OA is achieved.

FIG. 2 shows HA and HA-SH¹H NMR spectrum.

FIG. 3 is a representation of hydrogels of different formulations and their physicochemical and mechanical properties. (A) Preparing a hydrogel formula; (B) schematic representation of the hydrogel transition from solution to gel; (C) the transition time of the hydrogel from solution to gel; n is 3 and p<0.01, ns denotes p>0.05; (D) swelling ratio (Q) of hydrogel_m)，n＝3，* p<0.05，***p<0.001; (E) the hydrogel was incubated at 37 ℃ in 50U mL^-1In vitro degradation profiles in HA enzyme in PBS (top) and HA enzyme free PBS (bottom)Line, n ═ 3; (F) the upper graph is a scanning curve graph of storage modulus (G') versus frequency of hydrogel, and the control stress is 1 Pa; the lower graph shows the quantitative analysis of the G' and loss modulus (G ") of the hydrogel, n-3, p<0.01， ****p<0.0001。

FIG. 4 shows the shear-thinning behavior of 2-HA-Col hydrogels at 37 ℃.

FIG. 5 is an identification of ADSCs. (A) Morphology of ADSCs (Dio-stained cell membranes, green; Hochests-stained nuclei, blue). Scale bar 200 μm; (B, C) flow cytometry identification of surface biomarker of ADSCs as CD45^-And CD90⁺(ii) a And (D-F) identifying the multidirectional differentiation capability of the ADSCs, and respectively identifying the differentiation states of the ADSCs to adipogenic (D), osteogenic (E) and chondrogenic (F) lineages by oil red O, alizarin red and alcian blue staining, wherein the scale bar is 200 mu m.

FIG. 6 shows that the HA-Col hydrogel can promote cell proliferation and HAs good cell compatibility. (A) The morphology of ADSCs encapsulated in hydrogels of different formulations on

days

1, 3 and 5 of cell culture; (B) live/dead staining of ADSCs encapsulated in 2-HA-Col 3D reconstructed images after 3 days of culture (green for live cells, red for dead cells); (C and D) SEM images of 2-HA-Col hydrogels (red arrows indicate cells); (E and F) live/dead stained 3D reconstructed images of ADSCs cultured in 2-HA-Col hydrogel (left) and PBS (right) (E) and survival quantification statistics (F), n-3, p < 0.0001.

FIG. 7 is a 2D image of live/dead staining of ADSCs after 3 days of culture in 2-HA-Col hydrogel. Scale bar 200 μm.

Fig. 8 is a characterization of each nanoparticle. (A) Agarose gel electrophoresis bands of nanoparticles of different formulations; (B and C) Zeta potential (B) and particle size (C) of nanoparticles with different formulas, wherein n is 3.

FIG. 9 is a screening of nanoparticle formulations for transfection of ADSCs. (A) After transfecting ADSCs by using PBAE/plasmid DNA (pEGFP-TGF-beta 1) nanoparticles (with the mass ratio of 60/1, 90/1 and 120/1 respectively), the fluorescence expression (upper) and the cell morphology (lower) of EGFP in cells are scaled to 200 mu m; (B) flow cytometry analysis results show that the percentage of EGFP-positive cells increases with increasing mass ratio of PBAE/plasmid DNA nanoparticles; (C) the cytotoxicity of each group of nanoparticles was evaluated, n ═ 3, p <0.05, p < 0.001.

FIG. 10 shows the results of the preparation, characterization and co-culture experiments of T-ADSCs. (A) After the ADSCs are transfected by PBAE and plasmid DNA (pEGFP-TGF-beta 1) nanoparticles (the mass ratio is 60/1), the expression fluorescence of EGFP in cells is imaged, and the scale bar is 200 mu m; (B) percentage of EGFP positive cells, n-3; (C) quantifying TGF- β 1 concentration in culture supernatants of ADSCs and T-ADSCs by ELISA at different time points, n ═ 3, × <0.05, × < 0.01; (D) after PBAE/plasmid DNA nanoparticles (the mass ratio is 60/1) are transfected, cell proliferation of ADSCs is tested, n is 3, and ns represents p is more than 0.05; (E) the co-culture system is shown schematically, chondrocytes, ADSCs or T-ADSCs are cultured on the upper layer, and chondrocytes are cultured on the lower layer; (F) on day 3 of co-culture, qRT-PCR analysis of total RNA extracted from the lower cultured chondrocytes, n-3,. p <0.05,. p <0.01,. p < 0.001.

Figure 11 shows the procedure of surgical modeling of rats.

FIG. 12 shows the results of Micro-CT analysis. (A) Animal experiment design schematic diagram; (B and C) femur cross-sectional view (B, red arrow indicates cartilage damage) and joint longitudinal sectional view (C, red arrow indicates cartilage damage) of normal rats after treatment with PBS, ADSCs, T-ADSCs, gel + ADSCs or gel + T-ADSCs; (D) 3D reconstruction of knee joint micro-CT scan data set (red arrow indicates cartilage damage); (E) micro-CT imaging of trabecular bone microstructure parameter analysis of the representative region; (F and G) subchondral bone BV/TV and Tb.N quantification of tibia (F) and femur (G). n is 3, p <0.05, p < 0.01.

Figure 13 shows that T-ADSCs delivered via hydrogel significantly reduced the expression of TNF-a. (A) (ii) analysis of TNF- α concentration in synovial joint fluid of normal rats after treatment with PBS, ADSCs, T-ADSCs, gel + ADSCs or gel + T-ADSCs, n-3, p <0.05, p < 0.01; (B) h & E staining of synovial tissue of joints in each treatment group, 10 ×, scale bar 200 μm; 40X, scale bar 40 μm.

Fig. 14 shows the results of histological evaluation. (A) The pictures show the topography of the femoral surface cartilage for each treatment group (red boxes); (B and C) safranin O/fast green staining (B) and Col II immunohistochemical staining (C, red arrows indicate cartilage damage) of articular bone tissue of each treatment group, with a 10 × image scale of 200 μm; the 20 x image scale is 100 μm.

FIG. 15 is a schematic diagram of the pEGFP-TGF-. beta.1 plasmid.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result, have provided a composition for delivering genetically engineered ADSCs through an injectable hydrogel and its use for treating OA through a number of screens and tests. The invention unexpectedly discovers that compared with ADSCs, T-ADSCs can effectively inhibit inflammation and reduce cartilage and subchondral bone injury, and have more excellent OA treatment effect. The invention also provides an ECM-like hydrogel based on HA and Col I as a carrier for the intra-articular delivery of T-ADSCs, which can further enhance the curative effect of the T-ADSCs on OA. Experiments have shown that gel-delivered T-ADSCs show better anti-inflammatory and bone protective effects than other treatment groups. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "room temperature" or "ambient temperature" means a temperature of 4-40 ℃, preferably, 25 ± 5 ℃.

Osteoarthritis

Osteoarthritis (OA) is a chronic joint disease characterized by degeneration of articular cartilage and secondary hyperosteogeny. Also called osteoarthropathy, degenerative arthritis, senile arthritis, hypertrophic arthritis, etc.

The pathological features of OA are: degenerative damage to articular cartilage, subchondral bone sclerosis or cystic changes, joint marginal hyperostosis, synovial inflammation, synovial hyperplasia, joint capsule contracture, ligament relaxation or contracture, muscular atrophy and weakness, and the like. The clinical manifestations are slowly developing joint pain, tenderness, stiffness, joint swelling, limited mobility and joint deformity. This disease is commonly seen in middle-aged and elderly people, and is better in the heavy joints and joints with more physical activity, such as: knee, spine (cervical and lumbar), hip, ankle, hand, etc.

OA includes primary OA and secondary OA.

Transforming growth factor-beta 1

Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotropic, pleiotropic cytokine that regulates cell proliferation, differentiation, and apoptosis through receptor signaling pathways on the cell surface. Has important regulation effect on the synthesis of extracellular matrix, the repair of wound, immune function and the like. As used herein, the term "TGF- β 1" may refer to rat TGF- β 1 or an active fragment thereof as defined in SEQ ID NO 9, to human TGF- β 1 or an active fragment thereof, and to other species, preferably mammals, such as mice. As used herein, a biologically active fragment is meant to be a polypeptide that is a portion of a full-length TGF- β 1 polypeptide and yet retains all or part of the functionality of the full-length polypeptide. Typically, the biologically active fragment retains at least 50% of the activity of the full-length polypeptide. More preferably, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length polypeptide.

In another preferred embodiment, the TGF- β 1 is selected from human, rat or mouse TGF- β 1, and/or from an amino acid sequence having at least 80%, 90%, 95% or 99% identity to a human, rat or mouse TGF- β 1 amino acid sequence. Wherein the TGF-beta 1 amino acid sequence of human, rat and mouse is known to those skilled in the art or can be obtained by conventional sequencing methods.

Genetically engineered ADSCs overexpressing TGF-beta 1

As used herein, the terms "genetically engineered ADSCs overexpressing TGF- β 1", "T-ADSCs", "TGF- β 1 genetically engineered ADSCs", "genetically engineered adipose stem cells overexpressing transforming growth factor- β 1" are used interchangeably and refer to ADSCs that are capable of overexpressing TGF- β 1 after modification by engineering.

In the present invention, the ADSCs used to prepare the T-ADSCs are not particularly required and may be autologous, allogeneic, xenogeneic, or a combination thereof. Typically, the ADSCs are derived from mammals, such as humans, rats, mice.

As used herein, the term "overexpression" refers to an increase in TGF- β 1 expression by 20% or more, 30% or more, 40% or more, 50% or more, 80% or more, 100% or more, or even 200% or more, in cumulative expression, by the T-ADSCs compared to the ADSCs prior to genetic engineering, over the same experimental conditions, over the same time period or cell survival time period. It should be noted that the TGF-beta 1 expressed by the T-ADSCs and the TGF-beta 1 expressed by the ADSCs forming the T-ADSCs can be the same or different, and when the TGF-beta 1 expressed by the T-ADSCs is different, the TGF-beta 1 expressed by the T-ADSCs should be calculated by the sum of the TGF-beta 1 of different varieties produced by the T-ADSCs.

The method for preparing the T-ADSCs is not particularly required, and the T-ADSCs can be prepared by adopting a genetic engineering method commonly used in the field, and the method is known to the person skilled in the art. For example, a commonly used overexpression plasmid vector and a gene segment for encoding TGF-beta 1 are used for constructing a TGF-beta 1 overexpression plasmid and further delivering the TGF-beta 1 overexpression plasmid into cells (liposome, nanoparticle and the like can be used as delivery vectors), and a lentivirus overexpression plasmid can also be constructed for integrating a gene for encoding TGF-beta 1 into the nuclear genome of ADSCs. TGF-. beta.1 overexpression plasmids can be constructed using methods conventional in the art or commercially available. In one embodiment, a TGF- β 1 overexpression plasmid is prepared using a vector such as pcDNA3.1(+) capable of expression in eukaryotic cells to carry a gene fragment encoding TGF- β 1 (e.g., SEQ ID NO: 10). In general, overexpression plasmids contain stronger promoters to facilitate overexpression of the gene of interest.

Preferably, the T-ADSCs are prepared by a process that ensures the cellular activity of the ADSCs, preferably, the cellular activity of the T-ADSCs is not less than 80%, preferably, not less than 90%, not less than 95%, or even not less than 98% compared to the ADSCs before transfection.

In one embodiment, the process for preparing the T-ADSCs may comprise: and (3) inoculating the ADSCs into a cell culture dish at a certain density, carrying out adherent culture for 24 hours, carrying out serum-free transfection for 4 hours, then replacing with a complete culture medium, and carrying out transfection for 24 hours to obtain the T-ADSCs.

In the invention, the T-ADSCs can be directly formed by genetic engineering, and can also be passage cells thereof, as long as the passage cells can also over-express TGF-beta 1.

The invention discovers for the first time that T-ADSCs have significantly more excellent effect of treating OA compared with ADSCs.

Preferably, the T-ADSCs are delivered via an injectable hydrogel carrier.

Injectable hydrogel carriers and pharmaceutical compositions and uses thereof

In the present invention, the T-ADSCs are delivered via injectable hydrogels. Typically, the T-ADSCs are dispersed in a hydrogel-forming system and encapsulated at the same time as the hydrogel is formed. The injectable hydrogel encapsulating the T-ADSCs can be administered to the affected area by injection or transplantation.

The injectable hydrogel provides a suitable 3D microenvironment for the actions of cell attachment, expansion, proliferation and the like, and compared with the direct injection of cells, the injectable hydrogel can remarkably prolong the survival time of the cells and maintain the activity of the cells, thereby improving the treatment effect of the cells on affected parts.

Compared with the T-ADSCs only, the T-ADSCs loaded in the hydrogel have obviously more excellent effect of treating OA. More specifically, it has been experimentally demonstrated that T-ADSCs, or pharmaceutical compositions comprising the same, can be used for (but are not limited to) (a) increasing bone volume, or reducing a decrease in bone volume; (b) increasing the bone volume/tissue volume (BV/TV) ratio; (c) increasing the number of trabeculae, or decreasing the decrease in the number of trabeculae; (d) slowing down articular cartilage lesions; (e) slowing down the bone loss; (f) increasing bone density; (g) slowing down the decrease of aggrecan content in cartilage; (h) slowing down the reduction of the type II collagen content in the cartilage; (i) slowing the inflammatory response in the joint space; and/or (j) reducing TNF- α levels in joint synovial fluid.

In the present invention, there is no particular requirement for the selection of the injectable hydrogel carrier, and a hydrogel carrier suitable for in vivo use commonly used in the art may be selected as long as its toxicity, ability to promote cell adhesion and proliferation, ability to protect cells, degradability, and the like meet the requirements.

In another preferred embodiment, the hydrogel is composed of one or more materials selected from the group consisting of: polycarboxymethyl cellulose, alginates, hydroxypropylmethyl cellulose, carboxymethyl cellulose, ethyl hydroxyethyl cellulose, hydroxyalkyl cellulose, alkyl cellulose, polylactic acid, microcrystalline cellulose, polylactic-co-glycolic acid, dextrin, hydroxyethyl starch, hydroxyethyl chitosan, Hyaluronic Acid (HA), collagen, gelatin, and derivatives of HA, collagen, gelatin.

Preferably, the hydrogels are those commonly referred to as ECM-like hydrogels. Hyaluronic acid is one of the main components of joint synovial fluid, and HA in the ECM-like hydrogel can supplement the viscoelasticity of the joint synovial fluid and provide lubrication for joints, and HAs the effect of synergistically improving OA with T-ADSCs.

Preferably, the hydrogel is an HA-based and/or cross-linked HA hydrogel. Preferably, in addition to HA or cross-linked HA, the hydrogel further comprises one or more gel-modifying molecules (including, but not limited to) selected from the group consisting of: polycarboxymethyl cellulose, alginate, hydroxypropylmethyl cellulose, carboxymethyl cellulose, ethyl hydroxyethyl cellulose, hydroxyalkyl cellulose, alkyl cellulose, polylactic acid, microcrystalline cellulose, polylactic-co-glycolic acid, dextrin, hydroxyethyl starch, hydroxyethyl chitosan, collagen, gelatin, or a combination thereof.

Typically, the invention provides a self-assembly by Col I and thiol-containing group (e.g., -CH)₂CH₂SH) to obtain a hydrogel carrier.

Further, the hydrogel may also include one or more nutrients for the stem cells, including but not limited to: natural growth factors, salts, saccharides, protein components, nucleic acids, vitamins, etc., such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), L-glutamine, VC, VB, VE, or combinations thereof.

The precise amount of T-ADSCs that is provided to an individual in a therapeutically effective amount will depend on the mode of administration, the type and severity of the disease and/or condition, and the characteristics of the individual, such as general health, age, sex, weight, and tolerance to drugs. One of ordinary skill in the art will be able to determine the appropriate dosage based on these and other factors. When administered in combination with other therapeutic agents, the "therapeutically effective amount" of any other therapeutic agent will depend on the type of drug used. Suitable dosages are known for approved therapeutics.

Examples of subjects to which the pharmaceutical composition or therapeutic agent of the present invention is administered include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.).

As used herein, the terms "prevention" or "treatment" include disease-modifying treatment and symptomatic treatment, either of which may be prophylactic (i.e., to prevent, delay or lessen the severity of a symptom prior to the onset of the symptom)) or therapeutic (i.e., to lessen the severity and/or duration of a symptom after the onset of the symptom). "prevention" and "treatment" as used herein include delaying and stopping the progression of the disease, and do not require 100% inhibition, eradication, or reversal. In some embodiments, the reduction, alleviation, prevention, inhibition, and/or reversal is, e.g., at least about 1%, 10%, at least about 30%, at least about 50%, or at least about 80% as compared to the absence of the T-ADSCs, hydrogels, or pharmaceutical compositions comprising the T-ADSCs of the present invention.

Pharmaceutical composition and application

The present invention provides a pharmaceutical composition comprising: (a) genetically engineered modified ADSCs; and (b) an injectable hydrogel carrier encapsulating the genetically engineered ADSCs, wherein the genetically engineered ADSCs are T-ADSCs.

The OA is primary OA or secondary OA.

The invention discovers that the effect of the T-ADSCs on treating the OA can be fully exerted by delivering the T-ADSCs to the affected part through the hydrogel carrier, thereby realizing the maximization of the OA treatment effect. Experiments prove that the composition disclosed by the invention not only can relieve inflammation and protect cartilage, but also has an obvious protective effect on subchondral bone.

More specifically, the prevention and/or treatment of osteoarthritis comprises one or more characteristics selected from the group consisting of:

(a) increasing bone volume, or decreasing decrease in bone volume;

(b) increasing the bone volume/tissue volume (BV/TV) ratio;

(c) increasing trabecular bone number (tb.n), or decreasing the decline in trabecular bone number;

(d) slowing down articular cartilage lesions;

(e) slowing down the bone loss;

(f) increasing bone density;

(g) slowing down the decrease of aggrecan content in cartilage;

(i) slowing the inflammatory response in the joint space; and/or

(j) Reducing TNF-alpha levels in joint synovial fluid.

In another preferred embodiment, the pharmaceutical composition further comprises a second therapeutic agent including, but not limited to: anti-osteoporosis drugs (such as bisphosphonates (Clodronate, osteochosphine), Pamidronate (Pamidronate) and Tiludronate (Tiludronate)), Alendronate (Alendronate, Formoset, Fosamax) and Risedronate (Risedronate), Ibandronate (Boniva), Zoledronic acid (Reclean)), parathyroid hormone, prostaglandin E2, estrogens, estrogen receptor modulators, strontium salts (such as strontium ranelate), non-steroidal anti-inflammatory drugs (acetaminophen, diclofenac sodium, ibuprofen sustained release capsules, meloxicam), triamcinolone acetonide, tofacitinib, anti-inflammatory analgesics, antibacterial drugs, antiviral drugs, or combinations thereof.

The main advantages of the invention include:

the invention utilizes injectable hydrogel materials to deliver T-ADSCs, and has the following outstanding advantages:

(1) the ECM-like hydrogel has the advantages of promoting cell adhesion, adjustable physical and chemical properties and mechanical properties and the like, and the reticular structure of the ECM-like hydrogel can realize the exchange and circulation of oxygen, nutrient substances, metabolic waste and soluble factors and provide a proper 3D microenvironment for the actions of adhesion, expansion, proliferation and the like of ADSCs;

(2) the hydrogel can be injected and degraded, and realizes minimally invasive and safe cell transplantation;

(3) TGF-beta 1 protein has the defects of instability, quick degradation, short action time and poor drug-forming property; moreover, TGF-beta 1 protein is expensive. The active TGF-beta 1 is continuously generated and secreted by the ADSCs modified by genetic engineering, so that the local continuous delivery of the active TGF-beta 1 becomes possible, and the clinical application difficulty of directly administering the TGF-beta 1 is solved.

(4) The invention discovers for the first time that T-ADSCs can effectively inhibit inflammation and reduce bone injury compared with ADSCs, and has better effect of treating OA.

(5) The ADSCs modified by genetic engineering described in the invention can over-express and continuously secrete active TGF-beta 1, and the ADSCs can secrete other various chondroprotective and anti-inflammatory mediators. The combination of the two provides a more effective treatment means for treating OA.

Accordingly, the present invention provides the art with safe and effective drugs and methods for treating OA.

The invention is further illustrated by specific implementations. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Materials: HA (Mw 100kDa) was purchased from Bloomage Freda Biopharm co., Ltd. (shandong, china). 1. 4-butanediol diacrylate, 4-amino-1-butanol and 1- (3-aminopropyl) -4-methylpiperazine were purchased from Sigma-Aldrich (Shanghai, China). Other chemicals were purchased from alatin (shanghai, china). DMEM/F12 medium, Fetal Bovine Serum (FBS), penicillin and streptomycin were purchased from Gibco (Shanghai, China). Adipogenic, osteogenic and chondrogenic induced differentiation media for rat adipose-derived mesenchymal stem cells were purchased from seiko biotechnology limited (suzhou, china). CD90 and CD45 antibodies were purchased from BioLegend (san diego, california). Col II antibody, safranin O/fast green staining solution and H & E staining solution were purchased from Sauvir (Wuhan, China).

Type I collagen: collagen I, Col I

Type II collagen: collagen II, Col II

Aggrecan: aggrecan, Agg

Extracellular matrix: extracellar matrix, ECM

Characterization method

The mechanical properties of the hydrogels were tested with a Kinexus ultra + rheometer (Malvern). The prepared hydrogel was placed on 25mm parallel plates with a gap of 500-750 μm and frequency-swept at 37 deg.C (controlled stress of 1 Pa). The linear viscosity (η) was measured in a time-scanning mode at 37 ℃ and the shear-thinning behavior of the HA-Col hydrogel was characterized.

For the swelling test, hydrogel samples (150 μ L, 8mm diameter, 1.5mm height) were immersed in 1 × PBS and incubated at 37 ℃ for 24 hours. The water on the surface of the washed gel was sucked off using filter paper, and the mass of the swollen hydrogel was precisely measured (W)_t). Hydrogel samples were freeze-dried and weighed to determine W₀. By comparing W_tAnd W₀To calculate the swelling ratio (Q)_m)：

Q_m＝W_t/W₀

For the degradation test, sets of hydrogel samples of similar size (150 μ L, 8mm diameter and 1.5mm height) were prepared in Teflon moulds. The hydrogel samples were lyophilized and recorded as initial weight W₀. Will be driedThe hydrogel discs were immersed in PBS (pH 7.4) or contained 50U mL^-1HA enzyme (assist in saint, china) in PBS (pH 7.4). Hydrogel circular samples were incubated at 37 ℃ for 200rpm min using a constant temperature shaker (Zhichu ZQZY-CF, China)^-1Is rotated at the speed of (1). At a predetermined time point, the hydrogel round sample was removed from the solution, washed, lyophilized and weighed to obtain W_t. Hydrogel weight loss was calculated according to the following formula:

weight loss (%) - (W)₀-W_t)/W₀]×100％

Isolation and characterization of ADSCs: isolating ADSCs from adipose tissue in rat inguinal region, culturing the ADSCs in DMEM/F12 medium containing 10% FBS and 1% double antibody at 37 deg.C and 5% CO₂In the environment of (2). When the cells reached 80% confluence, the morphology of the ADSCs was visualized by imaging with an inverted microscope (Olympus). Surface biomarkers (CD45 and CD90) of ADSCs were identified using flow cytometry (BD Fortessa). Differentiation potential of ADSCs is identified through adipogenesis, osteogenesis and chondrogenesis induction experiments. All experiments used passage 3 ADSCs.

Isolation and characterization of chondrocytes: primary chondrocytes were isolated from rat articular cartilage, and cultured at 37 ℃ in DMEM/F12 medium containing 10% FBS and 1% double antibody at 5% CO₂In the environment of (2). Chondrocytes were identified by immunohistochemistry for type II collagen. Passage 1 chondrocytes were used in all experiments.

3D culture and cell injection experiments: suspending ADSCs in mixed solution of HA-SH and Col I to obtain cell concentration of 1 × 10⁶mL^-1The cell-hydrogel composition of (1). The cell-hydrogel compositions were transferred to 24-well plates and incubated at 37 ℃ and after 30min, 1mL of DMEM/F12 complete medium was added to each well. At predetermined time points, the morphology of the ADSCs in the different cell-hydrogel compositions was observed using a microscope and photographed. After 3 days, the selected formulations were evaluated for survival of ADSCs by live/dead stain analysis and photographed under a fluorescent microscope (Olympus). Evaluation of the protective Effect of the hydrogel on cells by cell injection experiment, 37 ℃ and 5% CO₂Under the conditions of (a) cell-hydrogel compositionIncubate in medium, then live/dead stain. Observations and photographs were taken under a confocal microscope (Leica) using ADSCs suspended in 1 x PBS as a control. Cell viability was calculated using ImageJ software.

And (4) SEM characterization: the microstructure of the hydrogel and the morphology of the cells in the hydrogel were observed using SEM (MIRA3, TESCAN). In the preparation of samples, a 5nm layer of gold (EM ACE600, Leica) was sprayed onto a cross section of the dried hydrogel or cell-hydrogel composition.

Preparation of T-ADSCs and optimization of transfection conditions: the optimal mass ratio of PBAE to pEGFP-TGF-. beta.1 plasmid DNA (GENEWIZ, Shanghai, China) was determined by agarose gel electrophoresis. The average particle size, particle size distribution and Zeta potential of the PBAE/plasmid DNA nanoparticles were measured using a dynamic light scattering spectrophotometer. To optimize transfection conditions, ADSCs were seeded into 24-well plates (6X 10)⁴Individual cells/well). After 24 hours, the medium was replaced with serum-free DMEM/F12 and cells were transfected with PBAE/plasmid DNA nanoparticles containing different mass ratios (2 μ g plasmid DNA/well). After 4 hours, the solution was changed. After 24 hours, the expression of EGFP in the cells was observed under a fluorescent microscope and the transfection efficiency was determined using a flow cytometer. The CCK-8 kit (bi yun, china) was used to assess the cytotoxicity and cell proliferation of the nanoparticles of different formulations.

Evaluation of expression of TGF-. beta.1 by T-ADSCs: ADSCs (2.5X 10)⁴Individual cells/well) were seeded in 24-well plates. After 12 hours, cells were transfected, expression of EGFP in the cells was observed under a fluorescent microscope, and the proportion of EGFP-positive cells was quantified using a flow cytometer. The concentrations of TGF-beta 1 in the culture supernatants of ADSCs and T-ADSCs were quantitatively determined at different time points using ELISA (Xinbo Sheng, China).

Co-culturing T-ADSCs and chondrocytes: in co-culture experiments, T-ADSCs prepared by the above method were used. Chondrocytes, ADSCs or T-ADSCs (2.5X 10)⁴Individual cells/well) were seeded in the basket and chondrocytes (2.5 × 10)⁴Individual cells) were seeded in wells under the basket using 10ng mL^-1DMEM/F12 culture of IL-1 β. Chondrocytes cultured in DMEM/F12 medium without IL-1 beta addition (chondrocytes in both the basket and the well) were used as a pairAccording to a group (Control group). After 3 days, total RNA was extracted from chondrocytes in each set of well plates and the relative expression levels of TNF- α, Col II and aggrecan (Agg) mRNA in each set were analyzed using qRT-PCR. Table 1 lists the primer sequences for qRT-PCR using GAPDH as internal reference.

TABLE 1

Surgically induced rat OA model: SD male rats (180-200g, SLAC laboratory animals Co., Ltd., Shanghai) were housed in a pathogen-free environment at the animal center of Shanghai university of transportation, and all animal experimental procedures were in compliance with the guidelines of the animal ethics Committee of Shanghai university of transportation. Secondary OA was induced in the right knee of the rat by anterior cruciate ligament transection and meniscal resection surgery. The specific procedure was as follows, using 1% sodium pentobarbital (40mg kg)^-1) Rats were anesthetized. The right rear knee hair was shaved and disinfected with iodophor. Two incisions (0.5-1 cm) were made in the skin and joint capsule with a #11 scalpel until the patella could subluxate laterally, exposing the joint cavity. The anterior cruciate ligament was exposed and transected with a pair of microscissors, and the anterior cruciate ligament was confirmed to be cut using the anterior drawer test. Subsequently, the meniscus was partially cut with a #11 blade. After completion, the patella is repositioned. The joint capsule is sutured with 5-0 absorbable suture, and the skin is sutured with 4-0 ordinary suture. Rats were injected with intraperitoneal penicillin sodium to prevent surgical-related infections. After 4 weeks of surgery, the rats were randomized into 5 groups (n ═ 6) and the rats were injected intra-articularly into each of the following groups: PBS (40. mu.L), ADSCs (5X 10)³Individual cells/. mu.L PBS, 40. mu.L), T-ADSCs (5X 10)³Individual cells/. mu.L PBS, 40. mu.L), Gel + ADSCs (5X 10)³Individual cells/. mu.L of hydrogel fraction, 40. mu.L) and Gel + T-ADSCs (5X 10)³Individual cells/μ L hydrogel component liquid, 40 μ L). Animals not subjected to surgery molding served as a control group (Normal).

TNF- α quantification: after 4 weeks of joint cavity administration, rats were sacrificed and joint synovial fluid was extracted. The TNF-alpha concentration in the synovial fluid of the joints of each group of animals was quantitatively determined using ELISA (synephrine, china).

Micro-CT: groups of rats were scanned for knee joints (90kV, 0.07mA) using micro-CT (VENUS micro-CT, China Pan Sheng). The CT image dataset was reconstructed in 3D and the trabecular bone parameters of the subchondral bone were analyzed using bone imaging processing and analysis software (VENUS, PINGSENG, china).

Histology: the knee joints of each group of rats were decalcified in a decalcifying solution for 5 weeks. Decalcified bone tissue was embedded in paraffin, 5nm sections were prepared and stained with H & E and safranin O (4% w/v)/fast green (0.1% w/v). Col II expression in cartilage was observed using Col II immunohistochemical staining.

Statistical analysis: all values are expressed as mean ± standard deviation. Statistical comparison data were analyzed using student's t-test with GraphPad Prism 8. p <0.05 represents statistically significant. Denotes p <0.01, p <0.001 and p <0.0001, respectively. p >0.05 not significant (ns).

Example 1

Preparation and characterization of ECM-like hydrogels

This example utilizes self-crosslinking of HA-SH and self-assembly of Col I to prepare ECM-like hydrogels and characterize the physicochemical and mechanical properties of hydrogels of different formulations.

(1) Synthesis and characterization of HA-SH

HA (1g) was dissolved in 100mL deionized water (deionized water, DI-H)₂O) and adjusting the pH to about 5.5. N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (1.22g, EDC) was added to the resulting solution and reacted for 30 minutes. Subsequently, N-hydroxysuccinimide (0.73g, NHS) and cysteamine hydrochloride (1.16g) were added to the reaction mixture, and the reaction was continued for 4 hours at pH 4.8. The reaction product was washed with DI-H containing 0.1M NaCl₂Dialyzing in O (pH 3.5) for 48 hours, and then in DI-H₂Dialyzed against O (pH 3.5) for 24 hours. Finally, the purified product is lyophilized and weighed to obtain HA-SH. Using 400MHz¹HA-SH was structurally characterized by H NMR (Agilent).¹The H NMR results showed that new resonance peaks, representing-CH, were observed at 2.52ppm and 2.31ppm₂CH₂Methylene protons of SH groups (fig. 2). The mercapto concentration of the reaction product HA-SH is 3.98X 10 determined by the Ellman method^-4mol g^-1。

(2) Preparation and characterization of hydrogels of different formulations

A Col I solution (1.3mg mL) was prepared using ice-cold 1 XPBS^-1) And (3) solution. An amount of HA-SH (pH 8.0) was dissolved using 600 μ L of Col I solution or 1 × PBS. Finally, the mixed solution was transferred to cylindrical Teflon molds of different diameters (8mm or 24mm) to obtain sets of hydrogels of similar size (height ═ 1.5 mm). The hydrogel formulation and components for each group are shown in figure 3A. It was observed by the backflow test that all hydrogel components could complete the solution-gel transition within 6 minutes (fig. 3B and 3C). The faster solution-gel transition speed may help the ADSCs to be uniformly encapsulated in the hydrogel without aggregation.

Q_m＝W_t/W₀

Swelling ratio measurements showed that the swelling ratio of the HA-SH hydrogel decreased with increasing HA mass fraction, ranging from 50.2 to 76.2 (fig. 3D). The addition of Col I reduced the swelling ratio of the gel.

For the degradation test, sets of hydrogel samples of similar size (150 μ L, 8mm diameter and 1.5mm height) were prepared in Teflon moulds. The hydrogel samples were lyophilized and recorded as initial weight W₀. The dried hydrogel discs were immersed in PBS (pH 7.4) or contained 50U mL^-1HA enzyme (assist in saint, china) in PBS (pH 7.4). Hydrogel circular samples were incubated at 37 ℃ for 200rpm min using a constant temperature shaker (Zhichu ZQZY-CF, China)^-1Is rotated at the speed of (1). At a predetermined time point, the hydrogel round sample was removed from the solution, washed, lyophilized and weighed to obtain W_t. Hydrogel weight loss was calculated according to the following formula:

Wight loss(％)＝[(W₀-W_t)/W₀]×100％

as shown in FIG. 3E, after 4 days, the 2-HA hydrogel was completely degraded in PBS containing HA enzyme. It is worth mentioning that when the 2-HA-Col hydrogel is formed by incorporating Col I, the degradation rate is delayed. The same was observed in PBS without HA enzyme, with more than 60% of the 2-HA hydrogel degrading within day 6, while the amount of 2-HA-Col degradation was less than 60%. In conclusion, the ECM-like hydrogel prepared has biodegradability; incorporation of Col I can delay the degradation time of the gel and thereby prolong the residence time of the gel in the body.

The mechanical properties of the hydrogels were tested using a Kinexus ultra + rheometer (Malvern). The prepared hydrogel was placed on 25mm parallel plates with a gap of 500-750 μm and frequency-swept at 37 deg.C (controlled stress of 1 Pa). The linear viscosity (η) was measured in a time-scanning mode at 37 ℃ and the shear-thinning behavior of the HA-Col hydrogel was characterized. As shown in FIG. 3F, the hydrogel's G ' and G ' increased with increasing HA mass fraction, and the addition of Col I increased G ' and G '. In all tested groups, the 2-HA hydrogel was the weakest, with a G' value of 162.5Pa and a G "value of 2.8 Pa. The incorporation of Col I can significantly increase G 'and G' to 206.4Pa and 6.9Pa, which is probably due to the fact that the collagen fibers formed by Col I enhance the hydrogel network and improve the mechanical properties of the hydrogel. In addition, HA-Col exhibits shear thinning properties, in which viscosity decreases with increasing shear rate, which makes it an ideal injectable cell delivery vehicle (fig. 4).

Example 2

Verification of the cellular compatibility of hydrogels

In this example, the cell compatibility of the hydrogel is evaluated by 3D culture experiment and live/dead staining, and first, the ADSCs are isolated from adipose tissue in the inguinal region of rat, and the specific isolation and extraction steps are as follows: (1) in order to avoid the contamination of bacteria in the extraction process, surgical instruments are sterilized at high temperature in advance. The rats were sacrificed, the hairs in the groin area of the rats were removed with a razor, and the rats were immersed in 75% ethanol for 10 min; (2) the sterilized rat was placed in a clean bench, the inguinal region of the rat was cut open with surgical scissors to expose the subcutaneous fat pad, the adipose tissues were peeled off with a surgical blade and forceps, immersed in cold PBS, and washed 3 times. Removing lymph node, fascia and blood vessel from adipose tissue, and cutting adipose tissue with scissors to about 1mm³Transferring the small particles into a centrifuge tube; (3) adding 5mL collagenase type I (1.5mg/mL) into the centrifuge tube, digesting in water bath at 37 ℃ for 1h, shaking the tissue uniformly every 10min until the fat tissue particles are digested into paste; (4) adding 5mL of DMEM/F12 complete medium to stop digestion, centrifuging (1000rpm/min, 10min), discarding the supernatant and undigested tissues, adding 5mL of DMEM/F12 complete medium, blowing and mixing uniformly by using a pipette gun, passing through a 70-micron cell screen, and centrifuging again (1000rpm/min, 5 min); (5) the supernatant was discarded, 3mL of DMEM/F12 was added to complete the medium to resuspend the cell pellet at the bottom of the tube, and after counting, the cells isolated by extraction were seeded at a density of 10 ten thousand/mL in a culture flask. Culturing ADSCs at 37 deg.C and 5% CO₂In the environment of (2). When the cells reached 80% confluence, the morphology of the ADSCs was observed under an inverted microscope (Olympus) and photographed. Surface biomarkers (CD45 and CD90) of ADSCs were identified using flow cytometry (BD Fortessa). Differentiation potential of ADSCs is identified through adipogenesis, osteogenesis and chondrogenesis induction experiments. All experiments used passage 3 ADSCs. As shown in FIG. 5, the extracted cells are spindle-shaped and are CD90⁺(99.5%) and CD45^-(0.335%) and can differentiate into fat cells, osteoblasts and chondrocytes, has the typical characteristics of ADSCs, and can meet the requirements of therapeutic applications.

In a 3D environment, the mechanical strength and microstructure of the hydrogel can affect the interaction between cells and a gel matrix, and further affect the cell morphology, phenotype, function and the like. Unlike 2D culture, cells tend to spread and proliferate more within 3D matrices that are mechanically weak, and if the mechanical strength of the 3D matrices is too strong, the spreading and proliferation of cells is slowed down. In this example, HA-Col hydrogel 3D cells were used to culture cells, and the cellular compatibility of each formulation hydrogel was preliminarily evaluated by observing the growth of the cells in the gel, and the cell was screenedSelecting candidate hydrogels suitable for cell growth. The specific implementation steps are as follows: suspending ADSCs in mixed solution of HA-SH and Col I to obtain cell concentration of 1 × 10⁶mL^-1The cell-hydrogel composition of (1), transferring the cell-hydrogel composition into a 24-well plate and incubating at 37 ℃, after 30min, adding 1mL of DMEM/F12 complete medium to each well, and observing the morphology of the ADSCs in different cell-hydrogel compositions using a microscope and photographing at a predetermined time point. As shown in FIG. 6A, ADSCs are not able to expand in the 4-HA-Col hydrogel matrix after 3D culture for 1, 3, 5 days, but maintain spherical morphology and proliferate slowly. Whereas ADSCs in 2-HA-Col and 3-HA-Col hydrogel matrices started to spread by day 1. Over time, the cells proliferated significantly in the 2-HA-Col and 3-HA-Col hydrogel matrices, extending morphologically further and forming a connected network between the cells. Compared with 3-HA-Col and 4-HA-Col aquagels, the 2-HA-Col aquagel can promote the expansion and proliferation of ADSCs and is beneficial to the growth of cells. After 3 days of 3D culture in the hydrogel, the candidate hydrogels were further evaluated for cellular compatibility by live/dead stain analysis. As shown in FIGS. 6B and 7, the survival rate of the cells in the 2-HA-Col hydrogel matrix was high as observed by fluorescence microscopy (Olympus). Based on the above results, the 2-HA-Col hydrogel exhibited excellent cell compatibility, and was used as a carrier for intra-articular cavity delivery of cells.

The microstructure of the hydrogel and the morphology of the cells in the hydrogel were observed using SEM (MIRA3, TESCAN), and samples were prepared by spraying a 5nm layer of gold (EM ACE600, Leica) onto a section of the dried hydrogel or cell-hydrogel composition. As shown in fig. 6C, the hydrogel has a net-like porous structure, facilitating the circulation of metabolic waste, secreted factors, and required nutrients of the encapsulated cells. As shown in FIG. 6D, after 24 hours of 3D culture, the attachment and spreading (indicated by red arrows) of ADSCs within the 2-HA-Col matrix was consistent with the above 3D culture white light observation, further verifying the cytocompatibility of the 2-HA-Col hydrogel.

The mechanical force damage and lack of 3D support matrix during cell injection results in decreased cell viability, and therefore, this factThe examples further evaluate the protective and supportive effect of 2-HA-Col hydrogels on cells in cell delivery. The specific implementation steps are as follows: at 37 ℃ and 5% CO₂The cell-hydrogel composition after injection was incubated in a culture medium for 1 hour, then live/dead staining, observed and photographed under a confocal microscope (Leica), and cell survival was calculated using ads cs suspended in 1 × PBS as a control using ImageJ software. As shown in fig. 6E and 6F, the survival rate of ADSCs in hydrogel was over 96% compared to PBS. Experiments prove that the 2-HA-Col hydrogel with the shear thinning characteristic can protect ADSCs from being damaged by mechanical force in the injection process, provides support protection for the ADSCs after injection, and improves the survival rate of transplanted cells.

FIG. 7 shows that the cells survived well in the 2-HA-Col hydrogel with better spreading, consistent with the 3D observations of dead/live staining shown in FIG. 6B.

In conclusion, the 2-HA-Col hydrogel HAs excellent cell compatibility, injectability and cell protection characteristics, and is very suitable to be used as an intra-articular delivery carrier of ADSCs.

Example 3

Preparation and characterization of T-ADSCs

Firstly, the transfection efficiency and cytotoxicity of the nanoparticles are evaluated, and the optimal nanoparticles are screened out for the preparation and characterization of T-ADSCs. Synthesizing PBAE as a delivery vector of plasmid DNA, and specifically synthesizing the following steps: (1)1, 4-butanediol diacrylate (0.4g) and 4-amino-1-butanol (0.15g) were added to a 5mL round flask and stirred at room temperature for 1 min; (2) putting the flask into a preheated oil bath at 90 ℃, and stirring for 24 hours to obtain acrylate-terminated poly (4-amino-1-butanol-co-1, 4-butanediol diacrylate); (3) after the reaction, the polymer (0.5g) obtained in the previous step was dissolved in 2mL of Tetrahydrofuran (THF). 1- (3-aminopropyl) -4-methylpiperazine (0.154g) was added to a polymer/THF solution, and the resulting solution was stirred at room temperature for 2 h; (4) the end-capped polymer was precipitated with diethyl ether to remove unreacted 1- (3-aminopropyl) -4-methylpiperazine. Subsequently, the resulting polymer was washed with diethyl ether; (5) vacuum drying for at least 48h, mixingThe purified PBAE was stored cryogenically. Use of chemical structure¹H NMR and molecular weight using GPC.

Preparation and characterization of PBAE/plasmid DNA nanoparticles: PBAE was dissolved in DMSO to prepare a stock solution (50mg/mL) and stored at-20 ℃. Then, PBAE stock solution (50mg/mL) was diluted with sodium acetate solution (25mM, pH 5) to obtain PBAE dilution of a certain concentration, plasmid DNA stock solution was diluted with DEPC water to obtain plasmid DNA dilution of a certain concentration, PBAE dilutions of different concentrations were added to the plasmid DNA dilution, mixed gently, and incubated for 10min to obtain PBAE/plasmid DNA mass ratios of 30: 1. 60: 1. 90: 1. 120: 1. The appropriate mass ratio of PBAE to plasmid DNA was screened by agarose gel electrophoresis. As shown in fig. 8A, when the mass ratio of PBAE to plasmid DNA in the nanoparticle is equal to or greater than 60/1, the plasmid DNA is completely encapsulated. Next, the biochemical physical properties of nanoparticles having PBAE/plasmid DNA mass ratios of 60/1, 90/1, and 120/1 were characterized using a dynamic light scattering spectrophotometer. As shown in FIGS. 8B and 8C, the zeta potential of each group of nanoparticles was 0-10mV, and the particle size was 200-250 nm.

The optimal nanoparticles are screened through an ADSCs transfection experiment and a cytotoxicity experiment, and the specific implementation steps are as follows: seeding ADSCs into 24-well plates (6X 10)⁴Individual cells/well), after 24 hours, the culture medium was replaced with DMEM/F12 without serum, cells were transfected with PBAE/plasmid DNA nanoparticles containing different mass ratios (2 μ g plasmid DNA/well), after 4 hours, the solution was changed, after 24 hours, the expression of EGFP in the cells was observed under a fluorescence microscope, and the transfection efficiency was determined using a flow cytometer, and the cytotoxicity of nanoparticles of different formulations was evaluated using CCK-8. As shown in fig. 9A and 9B, the ADSCs after transfection of each group of nanoparticles expressed EGFP, and flow quantification showed that the percentage of EGFP positive cells increased with the increase of the PBAE/plasmid DNA mass ratio, however, when the PBAE/plasmid DNA mass ratio was 120/1, a few round dead cells appeared after transfection. As shown in fig. 9C, the cytotoxicity of the nanoparticles increased with the increase of the mass ratio, and when the mass ratio of PBAE/plasmid DNA was 60/1, the cell compatibility of the nanoparticles was significantly better than that of the other two groups of nanoparticles. Therefore, the efficiency of cell transfection is considered comprehensivelyAnd cytotoxicity, the ADSCs are transfected by nanoparticles with a PBAE/plasmid DNA mass ratio of 60/1 to obtain T-ADSCs.

The example further studies the expression of TGF-beta 1 by T-ADSCs, and the specific implementation steps are as follows: ADSCs (2.5X 10)⁴Individual cells/well) in a 24-well plate, after 12 hours, transfecting the cells, observing the expression of EGFP in the cells under a fluorescence microscope, quantifying the proportion of EGFP positive cells by using a flow cytometer, and quantitatively detecting the concentration of TGF-beta 1 in culture supernatants of ADSCs and T-ADSCs by using ELISA (Xinbo, China) at different time points. The expression of the EGFP gene on the plasmid DNA, which is the plasmid DNAPEGFP-TGF-. beta.1 used in this example, was demonstrated by linking the EGFP gene to the TGF-. beta.1 gene using a 2A peptide, a cleavable peptide. As shown in fig. 10A, the fluorescence intensity of EGFP was strongest in the cells 2 days after transfection and then gradually decreased, and the expression of EGFP in the cells was still observed on day 5. This phenomenon is consistent with the flow quantitative detection result, after 2 days of transfection, 52.8 +/-0.99% of EGFP positive cells are present, the proportion of EGFP positive cells is gradually reduced, and it is worth mentioning that 20% of EGFP positive ADSCs still remain on the 5 th day, which proves that the screened PBAE/plasmid DNA nanoparticles have good transfection effect on ADSCs (FIG. 10B). To further examine the expression amount of TGF-. beta.1 in T-ADSCs at different time points, the concentration of TGF-. beta.1 in the culture supernatant of T-ADSCs was determined by ELISA. As shown in FIG. 10C, the expression level of TGF-beta 1 in the culture supernatant of T-ADSCs is significantly higher than that of ADSCs group at 1, 2, 3 and 4 days after transfection, and the cumulative expression level of T-ADSCs is 53.1% higher than that of ADSCs within 5 days. Cell proliferation experiments show that the absorbance (OD) value of the T-ADSCs group at each time point has no significant difference with that of a control group (control), and the screened nanoparticles for preparing the T-ADSCs have good cell compatibility (figure 10D).

In conclusion, the PBAE/plasmid DNA (mass ratio 60/1) nanoparticles with good transfection efficiency and cell compatibility to ADSCs are optimized and screened out in the embodiment, and can be efficiently and safely used for preparing T-ADSCs. The prepared T-ADSCs can continuously express and secrete TGF-beta 1.

Example 4

Co-culture experiments and qRT-PCR quantification

This example investigated the paracrine protective effect of T-ADSCs on OA-like chondrocytes using the Transwell co-culture system (FIG. 10E). The specific implementation steps are as follows: first, primary chondrocytes were isolated from rat articular cartilage, and cultured at 37 ℃ in DMEM/F12 medium containing 10% FBS and 1% double antibody at 5% CO₂In the environment of (1), chondrocytes were used for all experiments. Then, using the method described in example 3, T-ADSCs were prepared by mixing chondrocytes, ADSCs or T-ADSCs (2.5X 10)⁴Individual cells/well) were seeded in the basket and chondrocytes (2.5 × 10)⁴Individual cells) were seeded in wells under the basket using 10ng mL^-1DMEM/F12 culture of IL-1 β (a key pro-inflammatory cytokine in the OA microenvironment). Chondrocytes cultured in DMEM/F12 medium without IL-1 β addition (chondrocytes in both the basket and the well) were used as a Control group (Control group). After 3 days, total RNA was extracted from chondrocytes in each set of well plates and the relative expression levels of TNF-. beta.0, Col II and Agg mRNA in each set were analyzed using qRT-PCR. Table 1 lists the primer sequences for qRT-PCR using GAPDH as internal reference. qRT-PCR quantification showed that the expression of Col II and Agg mRNA was significantly reduced by 94.0% and 96.0% in IL-1. beta.1 group chondrocytes, respectively, compared to the Control group (Control), while the expression of TNF-. alpha.mRNA was up-regulated by 166.3-fold (FIG. 10F). Compared with the IL-1 beta group, the expression levels of Col II and Agg mRNA of the chondrocytes of the IL-1 beta + ADSCs group have no significant difference, the paracrine effect of the ADSCs modified by the genetic engineering is enhanced, and the expression levels of the Col II and the Agg mRNA of the chondrocytes of the IL-1 beta + T-ADSCs group are 3.268 times and 1.934 times higher than those of the IL-1 beta group respectively. In addition, the TNF-alpha expression of chondrocytes of the IL-1 beta + T-ADSCs group is reduced by 83.06 percent, which is obviously superior to that of the IL-1 beta + ADSCs group (reduced by 69.56 percent). The results prove that the paracrine protective effect of the T-ADSCs on OA-like chondrocytes is enhanced, and support is provided for the in-vivo application of the T-ADSCs.

Example 5

In vivo studies

This example evaluates the OA treatment effect of ADSCs, T-ADSCs, gel-encapsulated ADSCs, and gel-encapsulated T-ADSCs on SD rat OA model. Making SD maleSex rats (200g, SLAC laboratory animals Co., Ltd, Shanghai) were housed in a pathogen-free environment at the animal center of Shanghai university of transportation, and all animal experimental procedures were in compliance with the guidelines of the animal ethics Committee of Shanghai university of transportation. Secondary OA was induced in the right knee of the rat by anterior cruciate ligament transection and meniscal resection surgery. The specific implementation procedure is shown in FIG. 11, using 1% sodium pentobarbital (40mg kg)^-1) Anesthetizing the rat, removing the right rear knee hair and disinfecting with iodophor; two incisions (0.5-1 cm) were made in the skin and joint capsule with a #11 scalpel until the patella could be subluxated laterally, exposing the joint cavity, exposing the anterior cruciate ligament and transecting with microscissors, confirming the anterior cruciate ligament was sheared using the anterior drawer test, and then, the meniscus was partially resected with a #11 blade. After completion, the patella is repositioned, the joint capsule is sutured with 5-0 absorbable sutures, and the skin is sutured with 4-0 plain sutures. Rats were injected with intraperitoneal penicillin sodium to prevent surgical-related infections. After 4 weeks of surgery, the rats were randomized into 5 groups (n ═ 6), and the rats were injected intra-articularly on the fourth and sixth weeks with the following groups: PBS (40. mu.L), ADSCs (5X 10)³Individual cells/. mu.L PBS, 40. mu.L), T-ADSCs (5X 10)³Individual cells/. mu.L PBS, 40. mu.L), Gel + ADSCs (5X 10)³Individual cells/. mu.L of hydrogel fraction, 40. mu.L) and Gel + T-ADSCs (5X 10)³Individual cells/μ L hydrogel fraction, 40 μ L), the anti-inflammatory and chondroprotective effects of each treatment group were evaluated (fig. 12A). Animals not subjected to surgery molding served as a control group (Normal).

After 4 weeks of the first joint cavity injection, each group of rats was scanned for knee joints using micro-CT (VENUS micro-CT, china prosperity) (90kV, 0.07 mA). The CT image dataset was reconstructed in 3D and the trabecular bone parameters of the subchondral bone were analyzed using bone imaging processing and analysis software (VENUS, PINGSENG, china). The 2D images showed significant damage to cartilage (shown by red arrows) in PBS treated rats (fig. 12B and 12C). micro-CT scan 3D reconstructed maps of rat joints for each treatment group also confirm the above observations that the cell or gel + cell treated group may be able to significantly reduce cartilage damage compared to the PBS treated group (fig. 12D). The above results indicate that ECM-like hydrogels can synergistically enhance the therapeutic effect of cells on OA.

OA affects not only the articular cartilage but also the micro-structure of the subchondral bone, resulting in a reduction of subchondral bone BV/TV and tb.n. Thus, this example further analyzed trabecular micro-CT scan images of the tibia and femur in the region of interest to assess the effect of each treatment group on subchondral bone microstructure (fig. 12E). As shown in fig. 12F and 12G, BV/TV decreased by 38.2% and 17.2% in tibia and femur, respectively, and tb.n decreased by 26.4% and 29.6% in tibia and femur, respectively, of the PBS-treated group rats, as compared to the Normal group (Normal), with significant bone loss. However, after cell or Gel + cell treatment, bone loss was alleviated, and tibial BV/TV of rats in the Gel + T-ADSCs treated group was 22.9% higher than that in the PBS group, and significantly higher than that in the T-ADSCs or Gel + ADSCs group. Quantitative results showed that the Gel + T-ADSCs treated group was 28.6% and 42.1% higher than the PBS group for Tb.N in rat tibia and femur, respectively. In all treatment groups, Gel + T-ADSCs have the optimal effect of slowing the subchondral bone loss.

In OA joints, proinflammatory cytokines, especially TNF- α, produced by chondrocytes, osteoblasts, monocytes and synovial tissue drive an inflammatory cascade within the joint, causing persistent pain in the joint and exacerbation of the OA condition. This example evaluates the anti-inflammatory effect of each treatment group by killing rats, extracting synovial fluid from joints, and quantitatively measuring the TNF-alpha concentration in synovial fluid of joints of each group of animals using ELISA (euphorbia humilis, china) after 4 weeks of administration by articular cavity injection. As shown in fig. 13A, both the cell or gel + cell treated group significantly reduced TNF-a concentration in rat joint synovial fluid compared to the PBS group. In each treatment group, the anti-inflammatory effect of the T-ADSCs is obviously better than that of the ADSCs, and compared with the PBS group, the concentration of TNF-alpha in joint synovial fluid of rats treated by the T-ADSCs is reduced by 44.6%. In addition, compared with the PBS group, after the Gel + T-ADSCs are treated, the concentration of TNF-alpha in rat joint synovial fluid is reduced by 49.4%, the TNF-alpha concentration is obviously superior to that of the Gel + ADSCs and ADSCs treated group, and the optimal anti-inflammatory effect is shown. H & E staining of the synovial tissue of joints also confirmed the above findings, as shown in fig. 13B, in rats, the synovial tissue of joints in PBS-treated group had a large amount of inflammatory cell infiltration compared to Normal group. However, after cell or gel + cell treatment, the infiltration of inflammatory cells in synovial tissue of rat joints was reduced, and the infiltration of inflammatory cells in the gel + T-ADSCs treated group was minimal, thereby having the strongest anti-inflammatory therapeutic effect.

This example also observed the morphology of femoral articular cartilage and the pathological changes of articular bone tissue, as shown in fig. 14A, the thickness of articular cartilage was significantly reduced and the damage was significant (shown in the red frame) in the PBS treated group of rats, showing typical OA-like cartilage pathological features. After treatment with ADSCs, T-ADSCs, gel-encapsulated ADSCs or gel-encapsulated T-ADSCs, cartilage lesions in OA rats are alleviated. Furthermore, the cartilage thickness in the gel + cell treated group was significantly thicker than in the cell treated group alone. The knee joints of each group of rats were decalcified in a decalcifying solution for 5 weeks. The decalcified bone tissue was then embedded in paraffin to make 5nm sections, which were stained with H & E and safranin O (4% w/v)/fast green (0.1% w/v). As shown in fig. 14B, the Agg (orange) content in the surface layer and even the middle portion of the articular cartilage of the PBS-treated rats was significantly reduced. While cartilage degradation was much less severe in the cell or gel + cell treated groups, these groups showed higher Agg content than the PBS treated group, as evidenced by darker stained areas and greater thickness. Among all the treatment groups, the Gel + T-ADSCs group was most deeply colored, and thus had the best effect of slowing down the decrease in Agg content in articular cartilage of OA rats. Immunohistochemical staining was used to further assess the expression of Col II (an important component of cartilage ECM content) in articular bone tissue. As shown in FIG. 14C, the Col II content in PBS-treated group was severely reduced (light staining), consistent with the results of safranin O/fast green staining, in all treatment groups, the staining of the articular cartilage superficial and central regions of the rats in Gel + T-ADSCs-treated group was significantly darker, and the staining area of the articular cartilage tissue was larger, showing the best effect of slowing down the decrease of Col II expression in the articular cartilage of the OA rats.

In conclusion, in vivo experimental results show that in each treatment group, the gel + T-ADSCs have optimal anti-inflammatory and articular cartilage protection effects, and the OA disease development is effectively slowed down. On a macroscopic level, as shown by a micro-CT scanning 3D reconstruction diagram of the joint and the appearance of the femoral articular cartilage, Gel + T-ADSCs obviously slow down articular cartilage lesion and bone loss. On a microscopic level, histological staining shows that the infiltration of inflammatory cells of the gel + T-ADSCs group is less, the concentration of TNF-alpha in joint synovial fluid is obviously reduced, the anti-inflammatory effect is better, and the contents of Agg and Col II in the articular cartilage matrix are higher. The excellent therapeutic effect of OA in gel + T-ADSCs can be attributed to the following points: (1) the ECM-like hydrogel provides protection and support to the cells during and after injection, thereby improving survival of transplanted cells; (2) the TGF-beta 1 genetically engineered ADSCs enhance the paracrine action of the ADSCs, so that inflammation and inflammatory-related cartilage cell degenerative change are effectively inhibited; (3) component HA in the ECM-like hydrogel can supplement the viscoelasticity of joint synovial fluid and provide lubrication to the joint, thereby alleviating OA symptoms.

3 conclusion

The invention develops a pharmaceutical composition of injectable ECM-like hydrogels delivering T-ADSCs and its use for treating OA. The hydrogel has excellent cell compatibility, and can provide protection and support for cells during and after injection.

In addition, the ADSCs can over-express TGF-beta 1 by a genetic engineering method. The co-culture results show that T-ADSCs exert enhanced paracrine effects on OA-like chondrocytes. After 4 weeks of treatment, injection of ECM-like hydrogels loaded with T-ADSCs reduced cartilage degenerative changes and outperformed the use of cell only or ADSCs encapsulated hydrogel groups, as indicated by micro-CT and histological analysis. In addition to cartilage, the pharmaceutical composition of the present invention has the effect of protecting subchondral bone, for example, significantly increasing tb.n of subchondral bone and slowing bone loss of subchondral bone.

Furthermore, the hydrogel group encapsulating T-ADSCs showed the best in vivo anti-inflammatory effect in all tested groups, as shown by H & E staining of joint synovial tissue. In summary, the present invention provides a safe and convenient strategy for synergistically improving the therapeutic effect of OA by delivering genetically engineered ADSCs via ECM-like hydrogels.

The TGF-beta 1 overexpression plasmid used in the embodiment of the invention is a pEGFP-TGF-beta 1 plasmid, the composition of which is shown in figure 15, and the related sequences are summarized in the following table 2:

TABLE 2

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai university of transportation

<120> composition of injectable hydrogel and genetically engineered cells and its application for treating OA

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Ala Ser Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro

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Leu Pro Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu

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Ser Thr Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile

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Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro

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Pro Ser Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu

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Ala Leu Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Asp

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Pro Glu Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg

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Val Leu Met Val Asp Arg Asn Asn Ala Ile Tyr Asp Lys Thr Lys Asp

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Ile Thr His Ser Ile Tyr Met Phe Phe Asn Thr Ser Asp Ile Arg Glu

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Ala Val Pro Glu Pro Pro Leu Leu Ser Arg Ala Glu Leu Arg Leu Gln

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Arg Phe Lys Ser Thr Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr

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Ser Asn Asn Ser Trp Arg Tyr Leu Gly Asn Arg Leu Leu Thr Pro Thr

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Trp Leu Asn Gln Gly Asp Gly Ile Gln Gly Phe Arg Phe Ser Ala His

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Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr

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Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala

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Ser Ala Ser Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile

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Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met

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cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

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gctagcatgc cgccctcggg gctgcggctg ctgccgcttc tgctcccact cccgtggctt 60

ctagtgctga cgcccgggag gccagccgcg ggactctcca cctgcaagac catcgacatg 120

gagctggtga aacggaagcg catcgaagcc atccgtggcc agatcctgtc caaactaagg 180

ctcgccagtc ccccgagcca gggggaggta ccgccgggcc cgctgcccga ggcggtgctc 240

gctttgtaca acagcacccg cgaccgggtg gcaggcgaga gcgctgaccc ggagcccgag 300

cccgaggcgg actactacgc caaagaagtc acccgcgtgc taatggtgga ccgcaacaac 360

gcaatctatg acaaaaccaa agacatcaca cacagtatat atatgttctt caatacgtca 420

gacattcggg aagcagtgcc agaaccccca ttgctgtccc gtgcagagct gcgcctgcag 480

agattcaagt caactgtgga gcaacacgta gaactctacc agaaatatag caacaattcc 540

tggcgttacc ttggtaaccg gctgctgacc cccactgata cgcctgagtg gctgtctttt 600

gacgtcactg gagttgtccg gcagtggctg aaccaaggag acggaataca gggctttcgc 660

ttcagtgctc actgctcttg tgacagcaaa gataatgtac tccacgtgga aatcaatggg 720

atcagtccca aacgtcgagg tgacctgggc accatccatg acatgaaccg acccttcctg 780

ctcctcatgg ccacccccct ggaaagggct caacacctgc acagctccag gcaccggaga 840

gccctggata ccaactactg cttcagctcc acagagaaga actgctgtgt acggcagctg 900

tacattgact ttaggaagga cctgggttgg aagtggatcc acgagcccaa gggctaccat 960

gccaacttct gtctggggcc ctgcccctac atttggagcc tggacacaca gtacagcaag 1020

gtccttgccc tctacaacca acacaacccg ggtgcttccg catcaccgtg ctgcgtgccg 1080

caggctttgg agccactgcc catcgtctac tacgtgggtc gcaagcccaa ggtggagcag 1140

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gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

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aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggaattc 720

tgactcgag 729

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca 60

tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 120

ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 180

gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 240

ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg 300

tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 360

gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 420

ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 480

cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 540

agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 600

aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 660

gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 720

aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 780

gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 840

gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 900

caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 960

attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 1020

cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 1080

agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt 1140

ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 1200

tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 1260

tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 1320

aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 1380

ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 1440

ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 1500

caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 1560

atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 1620

acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 1680

ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 1740

aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 1800

aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 1860

gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 1920

ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc 1980

caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 2040

aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 2100

cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 2160

cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 2220

catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 2280

atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 2340

agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 2400

tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 2460

aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 2520

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 2580

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 2640

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 2700

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 2760

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 2820

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 2880

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 2940

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 3000

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 3060

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 3120

tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 3180

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtttttttg tttgcaagca 3240

gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 3300

tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 3360

gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 3420

tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 3480

ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 3540

ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 3600

tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 3660

aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 3720

gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 3780

gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 3840

ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 3900

gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 3960

gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 4020

gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 4080

tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 4140

gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 4200

agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 4260

aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 4320

ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 4380

gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc 4438

Claims

1. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of osteoarthritis, wherein the pharmaceutical composition comprises:

(a) genetically engineered modified adipose stem cells (ADSCs); and

wherein the ADSCs modified by genetic engineering are genetically engineered adipose-derived stem cells (T-ADSCs) over-expressing transforming growth factor-beta 1.

2. The use of claim 1, wherein the osteoarthritis is selected from the group consisting of: primary osteoarthritis or secondary osteoarthritis.

3. The use according to claim 1, wherein the prevention and/or treatment of osteoarthritis comprises one or more characteristics selected from the group consisting of:

(a) increasing bone volume, or decreasing decrease in bone volume;

(b) increasing the bone volume/tissue volume (BV/TV) ratio;

(d) slowing down articular cartilage lesions;

(e) slowing down the bone loss;

(f) increasing bone density;

(g) slowing down the decrease of aggrecan content in cartilage;

(i) slowing the inflammatory response in the joint space; and/or

(j) Reducing TNF-alpha levels in joint synovial fluid.

4. A pharmaceutical composition, comprising:

(a) genetically engineered modified ADSCs; and

5. The pharmaceutical composition of claim 4, wherein the hydrogel is comprised of one or more materials selected from the group consisting of: polycarboxymethyl cellulose, alginate, hydroxypropyl methyl cellulose, carboxymethyl cellulose, ethyl hydroxyethyl cellulose, hydroxyalkyl cellulose, alkyl cellulose, polylactic acid, microcrystalline cellulose, polylactic-co-glycolic acid, dextrin, hydroxyethyl starch, hydroxyethyl chitosan, hyaluronic acid, collagen, gelatin, and derivatives of hyaluronic acid, collagen, gelatin.

6. The pharmaceutical composition of claim 4, wherein the hydrogel is prepared by self-assembly of type I collagen and self-crosslinking of thiol-containing HA.

7. The pharmaceutical composition of claim 4, wherein the genetically engineered ADSCs overexpressing TGF- β 1 are obtained by transfecting ADSCs with TGF- β 1 overexpressing plasmid DNA.

8. The pharmaceutical composition of claim 4, wherein the ADSCs used to prepare the genetically engineered ADSCs overexpressing TGF- β 1 are from: autologous, allogeneic, xenogeneic, or a combination thereof.

9. The application of the genetically engineered ADSCs in preventing and/or treating osteoarthritis is disclosed, wherein the genetically engineered ADSCs are genetically engineered ADSCs over-expressing TGF-beta 1.

10. The use according to claim 9, wherein the prevention and/or treatment of osteoarthritis comprises one or more characteristics selected from the group consisting of:

(a) increasing bone volume, or decreasing decrease in bone volume;

(b) increasing the bone volume/tissue volume (BV/TV) ratio;

(d) slowing down articular cartilage lesions;

(e) slowing down the bone loss;

(f) increasing bone density;

(g) slowing down the decrease of aggrecan content in cartilage;

(i) slowing the inflammatory response in the joint space; and/or

(j) Reducing TNF-alpha levels in joint synovial fluid.