CN114306743A

CN114306743A - Three-phase bionic sleeve support and preparation method thereof

Info

Publication number: CN114306743A
Application number: CN202111391827.6A
Authority: CN
Inventors: 吕红斌; 史强; 胡建中; 陈灿; 许琰; 段嵘杰; 寻金瑞
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-04-12
Anticipated expiration: 2041-11-19
Also published as: CN114306743B

Abstract

The invention discloses a three-phase bionic sleeve support, which comprises a bone sleeve support, a cartilage sleeve support and a tendon support, wherein the cartilage sleeve support is sleeved outside the tendon support, the bone sleeve support is sleeved outside the cartilage sleeve support, and the bone sleeve support and the cartilage sleeve support as well as the cartilage sleeve support and the tendon support are in tight fit. The preparation method comprises preparing bone sleeve scaffold, cartilage sleeve scaffold and tendon scaffold, subjecting each scaffold to decellularization treatment, constructing C-BMP-2 modified decellularized bone sleeve scaffold, C-TGF-beta 3 modified decellularized cartilage sleeve scaffold and C-GDF-7 modified decellularized tendon scaffold, and intussuscepting and combining to obtain the final product. The invention has the functional activity of inducing stem cells to generate osteogenic, chondrogenic and tenogenic differentiation, can accelerate the regeneration of tendon insertion structures in bone tracts and the tendon remodeling in joint cavities after ACL reconstruction operation, and realizes the early joint function recovery after ACL reconstruction operation.

Description

Three-phase bionic sleeve support and preparation method thereof

Technical Field

The invention belongs to the technical field of medical instruments, and particularly relates to a three-phase bionic sleeve support and a preparation method thereof.

Background

The knee joint plays an important role in human activities. Among the ligaments constituting the knee joint, the Anterior Cruciate Ligament (ACL) connects the femur and tibia of the knee joint, and restricts excessive Anterior movement of the tibia during flexion and extension movements of the knee joint, and is the most important ligament for maintaining the stability of the knee joint. Unfortunately, ACL damage can cause knee joint instability, resulting in overstressing of the cartilage articular surfaces and menisci, accelerating their erosive wear, and ultimately causing Osteoarthritis (OA). ACL rupture is reported to be common in sports activities such as torsion, semi-flexion, sudden stop, impact, etc. as: football, basketball, badminton, rugby, etc. Epidemiological data show: the annual incidence rates of ACL damage in USA, Norway and Denmark are 0.068%, 0.034% and 0.038%, respectively.

In China, the epidemiological reports of ACL damage are few, but the results of the investigation of China's active training athletes by the three hospitals in North China show that the total incidence rate of ACL damage of the active training athletes is 0.43%. Therefore, ACL injuries belong to common sports injuries, threaten the health of the majority of sports enthusiasts and professional athletes, and the treatment of ACL injuries is a major challenge for sports medicine and orthopedists. Clinically, reconstructive surgery is often required after ACL disruption to restore knee joint stability. Grafts for ACL reconstruction include: autogenous tendon, allogenic tendon, artificial ligament, etc. After reconstruction, the joint cavity of the graft remodels the new tendon tissue, and the Bone tract of the graft regenerates the Bone-tendon insertion (BTI) structure, so as to realize the function of reconstructing ligament to replace ACL and maintain the stability of knee joint. Histologically, the normal tendon insertion point is composed of bone, fibrocartilage and tendon tissues and presents a continuous gradual change structure, so that stress concentration is avoided, and normal conduction of mechanical load is guaranteed. Research shows that in the early stage of ACL reconstruction, poor moulding reconstruction of transplanted ligaments in joint cavities leads to structural disorder and poor mechanical strength of regenerated ligaments and is easy to tear, meanwhile, the bone tract is often filled with pure fibrous scar tissues, and a characteristic bone-fibrocartilage-tendon structure is lost, so that firm integration of a graft and the bone tract cannot be effectively guaranteed, and the graft is easy to loosen. The data show that the recrapting rate after ACL reconstruction reaches 10% -25%. In recent years, with the continuous improvement of surgery and rehabilitation, the curative effect of ACL reconstruction surgery is improved to a certain extent, but patients still need 2 years of recovery time and still have higher risk of laceration. Therefore, how to accelerate generation of gradient bone tendon insertion points in a bone canal and regeneration of tendon tissues in a joint cavity after ACL reconstruction and restore biological functions of reconstructed ACL becomes a research focus in the field of sports medicine.

The explosive development of tissue engineering and regenerative medicine has motivated clinicians and researchers to attempt to solve this problem by constructing ACL-reconstructed tissue engineering grafts. At present, various ACL reconstruction grafts such as allogeneic tendons, high polymer material artificial ligaments and the like are applied to clinic, but the following problems exist: 1) most of the existing grafts imitate a single-phase structure of tendon tissue, and the existing grafts lack a bone-fibrocartilage-tendon three-phase structure in a bone duct and cannot provide a supporting material for the regeneration of the bone-fibrocartilage-tendon three-phase structure in the bone duct; 2) the existing graft lacks the functional activity of inducing stem cells to generate osteogenesis, chondrogenesis and differentiation of adult tendons, and is not beneficial to the formation of new bone tendon stops in the bone passage and the regeneration of tendons in the joint cavity; 3) the graft prepared by the artificial synthetic material has various problems of poor in-vivo degradation, biocompatibility, poor healing of the material and a bone tunnel and the like to be solved.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, break through the single-phase structure of the traditional ACL graft, and provide a three-phase bionic sleeve stent and a preparation method for simulating the morphological structure, mineral distribution and mechanical characteristics of human bone, fibrocartilage and tendon tissues.

In order to solve the technical problems, the invention adopts the following technical scheme.

The utility model provides a bionical sleeve support of three-phase, includes bone sleeve support, cartilage sleeve support and tendon support, bone sleeve support with cartilage sleeve support is hollow cylinder type structure, the tendon support is cylinder type structure, tendon support's outside cover is equipped with cartilage sleeve support, cartilage sleeve support overcoat is equipped with bone sleeve support, between bone sleeve support and the cartilage sleeve support with be the tight fit between the tendon support.

Foretell bionic sleeve support of three-phase, it is preferred, bone sleeve support is the bone sleeve support of decellularization, cartilage sleeve support is the cartilage sleeve support of decellularization, tendon support is the tendon support of decellularization.

Preferably, the bone sleeve scaffold is a decellularized bone sleeve scaffold modified by C-BMP-2, the cartilage sleeve scaffold is a decellularized cartilage sleeve scaffold modified by C-TGF-beta 3, the tendon scaffold is a decellularized tendon scaffold modified by C-GDF-7, the C-BMP-2 is bone morphogenetic protein-2 with collagen binding property, the C-TGF-beta 3 is transforming growth factor-beta 3 with collagen binding property, and the C-GDF-7 is growth differentiation factor 7 with collagen binding property.

As a general technical concept, the present invention also provides a method for preparing a three-phase bionic sleeve stent, comprising the steps of:

s1, preparing a bone sleeve scaffold, a cartilage sleeve scaffold and a tendon scaffold: trimming canine lumbar vertebra tissues into a hollow sleeve structure to obtain a bone sleeve support, trimming canine floating rib cartilage tissues into a hollow sleeve structure to obtain a cartilage sleeve support, trimming canine achilles tendon tissues into a cylindrical structure to obtain a tendon support, wherein the inner diameter of the bone sleeve support is equal to the outer diameter of the cartilage sleeve support, and the inner diameter of the cartilage sleeve support is equal to the diameter of the tendon support;

s2, carrying out decellularization treatment on the bone sleeve scaffold, the cartilage sleeve scaffold and the tendon scaffold: cleaning the bone sleeve scaffold, the cartilage sleeve scaffold and the tendon scaffold in a phosphate buffer solution, then performing freeze-thaw cycle, rinsing with a double-resistant phosphate buffer solution, putting the rinsed solution into a nuclease solution for digestion, and driving the double-resistant phosphate buffer solution to rinse by using a negative pressure suction mode to obtain a decellularized bone sleeve scaffold, a decellularized cartilage sleeve scaffold and a decellularized tendon scaffold;

s3, constructing a C-BMP-2 modified acellular bone sleeve scaffold, a C-TGF-beta 3 modified acellular cartilage sleeve scaffold and a C-GDF-7 modified acellular tendon scaffold: loading C-BMP-2 on a decellularized bone sleeve scaffold, loading C-TGF-beta 3 on the decellularized cartilage sleeve scaffold, loading C-GDF-7 on the decellularized tendon scaffold to obtain the C-BMP-2 modified decellularized bone sleeve scaffold, the C-TGF-beta 3 modified decellularized cartilage sleeve scaffold and the C-GDF-7 modified decellularized tendon scaffold, enabling the obtained bone sleeve scaffold to have stem cell osteogenesis inductive performance, enabling the obtained cartilage sleeve scaffold to have stem cell chondrogenesis inductive performance, enabling the obtained decellularized tendon scaffold to have stem cell tenogenesis inductive performance and realizing the slow release of growth factors, wherein the C-BMP-2 is bone morphogenetic protein-2 with collagen binding property, the C-TGF-beta 3 is transforming growth factor-beta 3 with collagen binding property, the C-GDF-7 is a growth differentiation factor 7 with collagen binding property;

s4: assembling: intussusception is carried out on the decellularized bone sleeve scaffold loaded with the C-BMP-2, the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3 and the decellularized tendon scaffold loaded with the C-GDF-7 obtained in the step S3, so that the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3 is sleeved outside the decellularized tendon scaffold loaded with the C-TGF-beta 7, the decellularized cartilage sleeve scaffold loaded with the C-BMP-2 is sleeved outside the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3, the decellularized bone sleeve scaffold loaded with the C-BMP-2, the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3 and the decellularized tendon scaffold loaded with the C-GDF-7 are all in tight fit, obtaining the three-phase bionic sleeve support.

Preferably, in the step S3, the saturated loading capacity of the C-BMP-2 on the decellularized bone scaffold is 10 μmol/L to 12 μmol/L, the saturated loading capacity of the C-TGF- β 3 on the cellcartilage scaffold is 8 μmol/L to 10 μmol/L, and the saturated loading capacity of the C-GDF-7 on the decellularized tendon scaffold is 10 μmol/L to 12 μmol/L.

Preferably, in step S2, the number of times of washing with the phosphate buffer solution is 3, and each washing is performed for at least 30min, and/or the number of times of rinsing with the double-resistant phosphate buffer solution is 3, and each rinsing is performed for at least 30 min.

In the above preparation method of the three-phase bionic sleeve stent, preferably, in step S2, the temperature of the nuclease solution is 37 ℃, the nuclease solution is digested for at least 2 hours, and the nuclease solution is composed of 100 to 200U/mL deoxyribonuclease I and 80 to 120 μ g/mL ribonuclease a.

Preferably, in the step S2, the freeze-thaw cycle includes two steps of freezing in liquid nitrogen for 2min to 5min and thawing in a thermostatic water bath at 37 ℃ for 10min to 15min, and the number of the freeze-thaw cycles is 5.

In the above method for preparing a three-phase bionic sleeve stent, preferably, in step S3, the C-BMP-2 is BMP-2 having collagen binding property, the C-TGF- β 3 is TGF- β 3 having collagen binding property, the C-GDF-7 is GDF-7 having collagen binding property, and the C-BMP-2, the C-TGF- β 3 and the C-GDF-7 are purified by detai biosynthesis.

Preferably, in step S1, the inner diameter of the bone sleeve support is 3.8mm to 4.2mm, the outer diameter of the bone sleeve support is 4.3mm to 4.7mm, the length of the bone sleeve support is 18mm to 22mm, the inner diameter of the cartilage sleeve support is 3.3mm to 3.7mm, the outer diameter of the cartilage sleeve support is 3.8mm to 4.2mm, the length of the cartilage sleeve support is 18mm to 22mm, the diameter of the tendon support is 3.3mm to 3.7mm, and the length of the tendon support is 53mm to 57 mm.

Preferably, before the step S4, the decellularized bone scaffold loaded with C-BMP-2, the decellularized cartilage scaffold loaded with C-TGF- β 3, and the decellularized tendon scaffold loaded with C-GDF-7 obtained in the step S3 are lyophilized and sterilized, and stored at an ultra-low temperature of-60 ℃ to-80 ℃ for further use.

When the three-phase bionic sleeve support is used, the cartilage sleeve support needs to be sleeved at the position of a bone tunnel to be transplanted and is matched with the length of the bone tunnel.

In the present invention, the following English abbreviation means:

BMP-2: bone morphogenetic protein-2.

TGF-. beta.3: transforming growth factor-beta 3.

GDF-7: growth differentiation factor 7.

C-BMP-2: collagen binding properties bone morphogenetic protein-2.

C-TGF-. beta.3: the collagen binding properties transform growth factor-beta 3.

C-GDF-7: collagen binding properties growth differentiation factor 7.

The C-BMP-2, C-TGF-beta 3 and C-GDF-7 adopted by the invention can be purchased by companies, and are generally obtained by using an escherichia coli prokaryotic expression system by adopting a genetic engineering technology, and the process is approximately as follows: utilizing software Primer premier 5.05 to assist in designing and synthesizing primers, amplifying ORF sequences of BMP-2, TGF-beta 3 and GDF-7 from human cDNA, respectively adding Hind III and Xho I restriction sites at the upstream and downstream of a target fragment, extracting plasmids from DH5 alpha E.Coli carrying pET30a-CAP plasmid in the laboratory, respectively carrying Hind III and Xho I restriction sites at the downstream of a Collagen affinity region (Collagen-affinity peptide CAP), respectively connecting amplification products of BMP-2, TGF-beta 3 and GDF-7 with pET30a-CAP plasmid after Hind III and Xho I double restriction, respectively, constructing expression vectors containing CAP, namely pET30a-C-BMP-2, pET30 a-C-TGF-beta 3 and pET30a-C-GDF-7, and transferring the constructed fusion expression vectors BL 83 into 21 fusion expression vectors, BMP-2 with collagen binding property, TGF-beta 3 with collagen binding property and GDF-7 with collagen binding property are collected and purified for subsequent experiments, and natural BMP-2(BMP-2), natural TGF-beta 3 (TGF-beta 3) and natural GDF-7(GDF-7) can be obtained by expression in the same way.

Compared with the prior art, the invention has the advantages that:

(1) the invention sequentially sleeves a cartilage sleeve support and a bone sleeve support on the bone tunnel part to be placed at the two ends of the tendon support from inside to outside so as to form a three-phase bionic sleeve support as an ACL reconstruction graft and provide a support material for the regeneration of bone-cartilage-tendon tissues in the bone tunnel.

(2) The three-phase bionic sleeve stent has the functional activity of inducing stem cells to generate osteogenic, chondrogenic and tenogenic differentiation, and has the advantages of accelerating regeneration of an osseous tendon stop structure in an osseous tract after an ACL reconstruction operation and tendon remodeling in a joint cavity and realizing early joint function recovery after the ACL reconstruction operation. The invention respectively adheres BMP-2 with collagen binding property, TGF-beta 3 with collagen binding property and GDF-7 with collagen binding property to bone, cartilage and tendon parts on a decellularized three-phase sleeve scaffold in a regionalization way, and strengthens the induced effects of the 'regionalization' of the scaffold in bone formation, cartilage formation and tendon formation.

(3) The invention adopts a three-phase bionic sleeve to emphasize that the bone tract part of the graft should simulate the morphological structure, mineral distribution and mechanical properties of bone, fibrocartilage and tendon tissues to carry out bionic design, and the scaffold is prepared into a sleeve shape, so that the acellular time can be greatly reduced, the influence on cells is small, and collagen binding characteristic growth factors are conveniently loaded, so that the scaffold has the functional activity of inducing stem cells to generate osteogenesis, chondrogenesis and tenogenesis, and the characteristic bone-fibrocartilage-tendon tissues are sequentially and efficiently regenerated at the graft and the bone tract, and meanwhile, tendon tissues with good tensile mechanical strength are regenerated in a joint cavity.

(4) The invention drives the circular flow of the decellularization reagent by a negative pressure suction decellularization method, washes and sucks out cell substances in the tissue, and realizes the high-efficiency removal of cells and antigen substances in the tissue and the maximum retention of extracellular matrix. The invention adopts the designed novel negative pressure suction decellularization scheme (patent document publication number is CN111084904A) to prepare the decellularized decalcification bone sleeve stent, the decellularized cartilage sleeve stent and the decellularized tendon stent, so that the decellularization time is greatly shortened, the decellularization is more thorough, and the influence on cells is small.

(5) According to the invention, the functional active three-phase bionic sleeve graft is innovatively constructed by a soaking method with simple operation, the optimal concentration of the combination of the growth factor with the collagen combination characteristic and the acellular scaffold is calculated, the stable attachment and slow release of the growth factor with the collagen combination characteristic are realized, and the stem cell induction activity of the scaffold is improved.

Drawings

Fig. 1 is a schematic structural diagram of a three-phase bionic sleeve support in embodiment 1 of the present invention.

Fig. 2 is a flow chart of the preparation of the three-phase bionic sleeve stent of embodiment 1 of the present invention.

Fig. 3 is a photograph of a lumbar vertebral body, a cartilage of a floating rib and an achilles tendon tissue of a beagle, which are obtained in the method for preparing the three-phase bionic sleeve stent of embodiment 1 of the present invention, wherein a is the lumbar vertebral body of the beagle, B is the cartilage of the floating rib of the beagle, and C is the achilles tendon tissue of the beagle.

Fig. 4 is a size photograph of the bone sleeve scaffold, the cartilage sleeve scaffold and the tendon scaffold obtained in the method for preparing the three-phase bionic sleeve scaffold in embodiment 1 of the present invention.

FIG. 5 is a histological and light source view of the decellularized bone sleeve scaffold, the decellularized cartilage sleeve scaffold and the decellularized tendon scaffold in example 1 of the present invention. H & E staining of bone sleeve support, cartilage sleeve support and tendon support before and after decellularization, the scale is 100 μm, collagen and proteoglycan content distribution diagram of bone sleeve support, cartilage sleeve support and tendon support before and after decellularization, the scale is 25 μm, wherein, Native: before decellularization, acellular: after decellularization, H & E: eosin-hematoxylin, Optical: white tablets, Collagen: collagen, PGs proteoglycans, Bone: bone, Cartilage: cartilage, Tendon: tendons of the animal.

FIG. 6 is a scanning electron micrograph of the surface topology changes of the bone sleeve scaffold, the cartilage sleeve scaffold and the tendon scaffold before and after decellularization in example 1 of the present invention.

FIG. 7 is a scanning electron micrograph of Bone Mesenchymal Stem Cells (BMSCs) adhered to the surfaces of a decellularized bone sleeve scaffold, a cartilage sleeve scaffold and a tendon scaffold in example 1 of the present invention, with a scale of 20 μm.

FIG. 8 immunofluorescence of Calcein-AM/PI live and dead cell double staining for assessing the viability of BMSCs on the surface of decellularized bone, cartilage and tendon scaffolds in example 1 of the present invention, wherein the scale is 200 μm, Optical: white tablet, Calcein-AM: live cells, PI: dead cells, Merge: and (6) merging.

FIG. 9 is a schematic view of a novel decellularized three-phase sleeve scaffold having a regional differentiation induction function according to example 1 of the present invention. The bone sleeve is loaded with CBD-BMP-2, the cartilage sleeve is loaded with CBD-TGF beta, the tendon scaffold is loaded with CBD-GDF-7, and the internal biological activity of the decellularized three-phase sleeve scaffold is improved through the BMP-2, TGF-beta 3 and GDF-7 which are specifically combined by collagen.

FIG. 10 is a graph showing the binding of C-BMP-2, BMP-2 to a bone scaffold in example 1 of the present invention.

FIG. 11 is a graph showing the binding of C-TGF-. beta.3, TGF-. beta.3 and cartilage fragments in example 1 of the present invention.

FIG. 12 is a graph showing the binding of C-GDF-7, GDF-7 and tendon scaffold in example 1 of the present invention.

FIG. 13 is a graph showing the release kinetics of growth factors in the C-BMP-2 modified bone sleeve scaffold and the BMP-2 modified bone sleeve scaffold of example 1 of the present invention.

FIG. 14 is a graph showing the kinetics of growth factor release in the C-TGF-. beta.3-modified cartilage sleeve scaffold and the TGF-. beta.3-modified cartilage sleeve scaffold according to example 1 of the present invention.

FIG. 15 is the release kinetics curves of the C-GDF-7 modified tendon scaffold and the GDF-7 modified tendon scaffold in example 1 of the present invention.

FIG. 16 is a schematic view of the specimen at 4 weeks and 16 weeks after the operation in example 1 of the present invention. CTL: control group (autologous popliteal muscle graft group); SATS: experimental group (three-phase bionic sleeve bracket group); NAT: normal tissue group (Native).

FIG. 17 is a photograph showing the histomorphological evaluation at 4 weeks and 16 weeks after the operation in example 1 of the present invention, (A) H & E staining of sample lines at 4 weeks and 16 weeks after the operation. (B) Masson staining was performed on specimens 4 weeks and 16 weeks after surgery.

FIG. 18 is a graph showing histological evaluation of neonatal tendon interface at 4 weeks and 16 weeks after the operation in example 1 of the present invention by semi-quantitative analysis using image analysis software.

FIG. 19 is a graph showing the semi-quantitative analysis of the change in thickness of fibrocartilage at 4 weeks and 16 weeks using image analysis software in 4 weeks and 16 weeks after the operation of the imaging test in example 1 of the present invention.

FIG. 20 is a graph showing the semi-quantitative analysis of the change of optical density of cartilage matrix at 4 weeks and 16 weeks by image analysis software in 4 weeks and 16 weeks after the operation in example 1 of the present invention.

FIG. 21 is a line X-ray microscopic scan of 4 weeks and 16 weeks post-operative samples of example 1 of the present invention, wherein CTL: control group (autopopliteal cord graft group), sat: experimental group (function optimized three-phase bionic socket ACL graft group), NAT: normal group (Native).

FIG. 22 is a graph showing bone volume fraction (BV/TV) of two groups of specimens at 4 and 16 weeks after the quantitative analysis in example 1 of the present invention, and the dotted line represents the normal value of BV/TV.

Fig. 23 is a graph of trabecular bone thickness (tb.th) at 4 weeks and 16 weeks after the quantitative analysis in example 1 of the present invention, and the dotted line represents the tb.th normal value.

Fig. 24 is a graph showing the trabecular bone number (tb.n) of two groups of specimens at 4 weeks and 16 weeks after the quantitative analysis in example 1 of the present invention, where n is 6. The dashed line represents the tb.n normal value.

Fig. 25 is a graph for evaluating the influence of a three-phase bionic sleeve ACL graft on the restoration of mechanical properties in the ACL new-born tendon interface injury repair process in 4-week and 16-week postoperative biomechanical tests in example 1 of the present invention, where the test index is a tensile Load (Failure Load), and the dotted line represents a normal value of the tensile Load.

Fig. 26 is a graph of evaluation of the effect of a three-phase bionic socket ACL graft on the restoration of mechanical properties during the repair of ACL neogenetic bone-tendon interface injury in 4-week and 16-week postoperative biomechanical tests in example 1 of the present invention, where the test index is Stiffness (Stiffness), and the dotted line represents the normal value of Stiffness.

In the above figures, P <0.05, P <0.01, P <0.001, ns: p > 0.05.

Illustration of the drawings:

1. a bone sleeve support; 2. a cartilage sleeve support; 3. a tendon scaffold.

Detailed Description

The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention. The materials and equipment used in the following examples are commercially available.

Example 1

The invention discloses a three-phase bionic sleeve support, which comprises a bone sleeve support 1, a cartilage sleeve support 2 and a tendon support 3, wherein the bone sleeve support 1 and the cartilage sleeve support 2 are both hollow cylindrical structures, the tendon support 3 is a cylindrical structure, the cartilage sleeve support 2 is sleeved outside the tendon support 3, the bone sleeve support 1 is sleeved outside the cartilage sleeve support 2, and the bone sleeve support 1 is tightly matched with the cartilage sleeve support 2 and the cartilage sleeve support 2 is tightly matched with the tendon support 3.

In one embodiment of the present embodiment, the bone sleeve scaffold 1 is made of decellularized bone, the cartilage sleeve scaffold 2 is made of decellularized cartilage, and the tendon scaffold 3 is made of decellularized tendon. The bone sleeve support is a homogeneously mineralized bone sleeve support, the cartilage sleeve support is a gradient mineralized cartilage sleeve support, and the bone sleeve support, the cartilage sleeve support and the tendon support can be attached with an induced active exosome, so that stem cells are stimulated to generate specific differentiation through the induced active exosome, and the tissue regeneration capacity is improved.

Specifically, in this embodiment, the bone sleeve scaffold 1 is a C-BMP-2 modified decellularized bone sleeve scaffold, the cartilage sleeve scaffold 2 is a C-TGF- β 3 modified decellularized cartilage sleeve scaffold, the tendon scaffold 3 is a C-GDF-7 modified decellularized tendon scaffold, the C-BMP-2 is a bone morphogenetic protein-2 with collagen binding property, the C-TGF- β 3 is a transforming growth factor- β 3 with collagen binding property, and the C-GDF-7 is a growth differentiation factor 7 with collagen binding property.

A method for preparing a three-phase bionic sleeve stent of the embodiment, as shown in fig. 2, includes the following steps:

s1, preparing a bone sleeve bracket 1, a cartilage sleeve bracket 2 and a tendon bracket 3:

(1) obtaining lumbar vertebral bodies, cartilage of the floating ribs and achilles tendon tissues of beagle dogs:

taking a dog prone position, cutting the dorsal skin of the lumbar vertebra, removing muscles and periosteum, exposing the lumbar vertebra centrum, completely taking out lumbar vertebra centrum tissues after 3 sections of lumbar vertebra are cut off by using a saw, removing joint surfaces at two ends of the lumbar vertebra centrum after cleaning by using normal saline, and cleaning a marrow cavity to obtain a complete lumbar vertebra centrum as shown in figure 3;

taking a dog in a supine position, touching the rib area, longitudinally cutting about 6cm right above the dog, removing superficial fascia and muscle tissues, exposing a rib framework, clenching off the rib with rongeur forceps, separating the floating rib, repeatedly washing with normal saline, and trimming off membranous tissues on the surface with a sharp blade to obtain the floating rib tissues, as shown in fig. 3;

the dog is taken to lie on the stomach, after touching the achilles tendon part of the calf, the skin is cut along one side of the achilles tendon, the achilles tendon and the connected triceps surae are exposed and separated at the achilles bone stopping point and the triceps surae muscle moving part respectively, the muscles attached to the surface are removed, the achilles tendon tissue is separated by a sharp blade, and the single bundle of achilles tendon is obtained after being washed by normal saline, as shown in figure 3.

(2) Preparation of bone sleeve scaffolds (sleeve bone tissue): collected lumbar vertebraeThe bone tissue of the vertebral body is trimmed to 30 multiplied by 30mm³Sleeve-shaped bone tissue having an inner diameter of 4.0mm, an outer diameter of 4.5mm and a length of 20mm was prepared using a mini bench drill and a ring drill having diameters of 3.5mm and 4.0mm, respectively, as shown in fig. 4.

(3) Preparation of cartilage sleeve scaffolds (sleeve-like cartilage tissue): trimming collected cartilage tissue of canine floating rib into size of 30 × 15 × 15mm³Preparing sleeve-shaped cartilage tissue by using a mini bench drill and a ring drill with the diameter of 3.5mm and 4.0mm respectively: an inner diameter of 3.5mm, an outer diameter of 4.0mm and a length of 20mm, as shown in FIG. 4.

(4) Preparation of tendon scaffolds (columnar tendon tissue): collected canine Achilles tendon tissue was trimmed to a size of 60X 5mm³Preparing columnar tendon tissue by using a mini bench drill and an annular drill with the diameter of 3.5 mm: diameter 3.5mm and length 55mm as shown in figure 4.

S2, carrying out decellularization treatment on the bone sleeve support 1, the cartilage sleeve support 2 and the tendon support 3:

(1) respectively cleaning the bone sleeve stent 1, the cartilage sleeve stent 2 and the tendon stent 3 in Phosphate Buffered Saline (PBS) for three times, washing for 30min each time, and then wrapping with gauze;

(2) performing five freeze-thaw cycles on the bone sleeve scaffold 1, the cartilage sleeve scaffold 2 and the tendon scaffold 3, and then rinsing with 1% volume fraction of double-antibody PBS for three times, each time for 30 min; the freeze-thaw cycle here is: freezing in liquid nitrogen for 2min, and thawing in a constant-temperature water bath kettle at 37 deg.C for 10 min;

(3) placing the bone sleeve bracket 1, the cartilage sleeve bracket 2 and the tendon bracket 3 into a nuclease solution at 37 ℃ for digestion for 2 hours, wherein the ratio of the nuclease solution is 150U/mL deoxyribonuclease I +100 mug/mL ribonuclease A;

(4) 1% double-antibody PBS (phosphate buffer solution) is driven to wash for 3 hours by a negative pressure suction decellularization method for the bone sleeve bracket 1, the cartilage sleeve bracket 2 and the tendon bracket 3; the negative pressure aspiration decellularization method can be performed by using the method and apparatus disclosed in application No. 202010103858.6 and publication No. CN 111084904A. The histology and light source images, scanning electron microscope images before and after decellularization of the scaffold, scanning electron microscope images of scaffold cell adhesion, and double staining images of live and dead cells of Calcein-AM/PI are shown in FIGS. 5 to 8.

The PBS buffer, double-resistant PBS, DNase I, and RNAse A were all commercially available.

S3, constructing a C-BMP-2 modified bone sleeve scaffold, a C-TGF-beta 3 modified cartilage sleeve scaffold and a C-GDF-7 modified tendon scaffold: loading C-BMP-2 on a decellularized bone sleeve scaffold, loading C-TGF-beta 3 on the decellularized cartilage sleeve scaffold, loading C-GDF-7 on the decellularized tendon scaffold to obtain the C-BMP-2 modified decellularized bone sleeve scaffold, the C-TGF-beta 3 modified decellularized cartilage sleeve scaffold and the C-GDF-7 modified decellularized tendon scaffold, enabling the obtained bone sleeve scaffold to have stem cell osteogenesis inductive performance, enabling the obtained cartilage sleeve scaffold to have stem cell chondrogenesis inductive performance, enabling the obtained decellularized tendon scaffold to have stem cell tenogenesis inductive performance and realizing the slow release of growth factors, wherein the C-BMP-2 is bone morphogenetic protein-2 with collagen binding property, the C-TGF-beta 3 is transforming growth factor-beta 3 with collagen binding property, the C-GDF-7 is a growth differentiation factor 7 with collagen binding property; the decellularized bone sleeve scaffold loaded with C-BMP-2, the decellularized cartilage sleeve scaffold loaded with C-TGF-beta 3 and the decellularized tendon scaffold loaded with C-GDF-7 can be freeze-dried and sterilized, and are stored at the ultralow temperature of-60 to-80 ℃ for later use.

S4: assembling: intussusception is carried out on the decellularized bone sleeve scaffold loaded with the C-BMP-2, the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3 and the decellularized tendon scaffold loaded with the C-GDF-7 which are obtained in the step S3, the C-TGF-beta 3 modified cartilage sleeve scaffold and the C-BMP-2 modified bone sleeve scaffold are sequentially sleeved at two ends of the decellularized tendon scaffold modified by the C-GDF-7, the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3 and matched with the length of a bone tunnel are sleeved outside the decellularized tendon scaffold loaded with the C-GDF-7, the decellularized bone sleeve scaffold loaded with the C-BMP-2 and the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3 are sleeved outside the decellularized cartilage sleeve scaffold loaded with the C-TGF-beta 3, The acellular cartilage sleeve stent loaded with the C-TGF-beta 3 and the acellular tendon stent loaded with the C-GDF-7 are in tight fit to obtain a three-phase bionic sleeve stent, namely, a novel three-phase sleeve ACL graft is assembled, and the graft implanted into an animal body is subjected to freeze drying and ethylene oxide disinfection and then is sealed and stored.

In this embodiment, the research procedure of step S3 is as follows:

(1) preparing PBS solutions (0. mu.M, 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M and 12. mu.M) containing different concentrations of the binding factors C-BMP-2, C-TGF-beta 3 and C-GDF-7 and the natural factors BMP-2, TGF-beta 3 and GDF-7;

(2) the three-phase sleeve scaffold is divided into bone, cartilage and tendon single-phase scaffolds, and the weights of the three scaffolds are respectively 8.01 +/-0.22 mg, 1.19 +/-0.07 mg and 1.23 +/-0.10 mg. 3 48 pore plates are taken and respectively added into the bone sleeve bracket 1, the cartilage sleeve bracket 2 and the tendon bracket 3. Adding 200 mu L of solutions of C-BMP-2, C-TGF-beta 3, C-GDF-7, BMP-2, TGF-beta 3 and GDF-7 with different concentrations into each 1 hole respectively; incubating at 4 ℃ for 12 hours, sucking out the growth factor solution, and gently rinsing with PBS; anti-6 XHis tag antibody (anti-6 XHis tag antibody, 1: 1000, Sigma) was added, incubated at 37 ℃ for 1 hour, and gently rinsed with PBS; adding an AP-coupled anti-mouse IgG antibody (1: 10000, Sigma), incubating at 37 ℃ for 1 hour, gently rinsing by PBS, adding 4-nitrophenyl disodium phosphate (2mg/mL), reacting for 10 minutes, then stopping the reaction by 0.2M sodium hydroxide, measuring the absorbance of the solution by a 405nm waveband spectrophotometer (the absorbance is in positive correlation with the binding amount of factors on the scaffold), drawing a binding curve of the growth factors and the scaffold as shown in figures 9 to 12, determining the saturation load concentration of the collagen binding characteristic growth factors of the bone sleeve, the cartilage sleeve and the tendon part, and calculating the limit factor loading amount of the three scaffolds according to the concentration. The binding curves show: in the bone sleeve scaffold, BMP-2 reaches a saturation binding state at about 7-8 μ M, and C-BMP-2 reaches a saturation binding state at 10-12 μ M; at the same concentration, the binding capacity of C-BMP-2 on the bone sleeve bracket is obviously more than that of BMP-2. In the cartilage sleeve scaffold, TGF-beta 3 reaches a saturation binding state at about 6 mu M, and C-TGF-beta 3 reaches a saturation binding state at 8-10 mu M; at the same concentration, the amount of C-TGF-beta 3 bound to the cartilage sleeve scaffold was significantly greater than TGF-beta 3. In tendon part scaffold, GDF-7 reaches saturation binding state at about 8 μ M, and C-GDF-7 reaches saturation binding state at 10-12 μ M; at the same concentration, the amount of C-GDF-7 bound to the tendon scaffold was significantly greater than that of GDF-7. The results show that C-BMP-2, C-TGF-beta 3 and C-GDF-7 have stronger collagen binding capacity in the acellular scaffold than BMP-2, TGF-beta 3 and GDF-7.

(3) Placing three scaffolds (C-BMP-2 modified bone sleeve scaffold, C-TGF-beta 3 modified cartilage sleeve scaffold and C-GDF-7 modified tendon scaffold) with the same mass in a 48-pore plate, adding 200 muL PBS, simulating a body fluid environment, and horizontally shaking in a shaking table at 80rpm and 37 ℃; PBS was changed every 24 hours from shaking the rack until day 9; according to the above method, anti-6 XHis tag antibody (1: 1000, Sigma) was added to the scaffold, incubated at 37 ℃ for 1 hour, and gently rinsed with PBS; AP-conjugated anti-mouse IgG antibody (1: 10000, Sigma) was added, incubation was carried out at 37 ℃ for 1 hour, PBS was gently rinsed, 4-nitrophenyl disodium phosphate (2mg/mL) was added, reaction was terminated after 10 minutes by 0.2M sodium hydroxide, solution absorbance (absorbance represents the content of scaffold residual factor) was measured with a 405 nm-band spectrophotometer, and a growth factor release curve was plotted as shown in FIGS. 13 to 15.

The results of the scaffold growth factor release kinetics evaluation show that: the release speed of the growth factors on the combined type factor modified bracket group is slower than that of the natural type factor modified bracket group. BMP-2 persisted 3.64 + -0.71% on BMP-2 modified bone sleeve scaffolds on day 9, while BMP-2 persisted 34.54 + -2.23% on C-BMP-2 modified bone sleeve scaffolds; TGF-beta 3 remains 3.90 +/-0.53% on the TGF-beta 3 modified cartilage sleeve scaffold, and TGF-beta 3 remains 37.98 +/-2.54% on the C-TGF-beta 3 modified bone sleeve scaffold; GDF-7 remains 4.84 + -0.49% on the GDF-7 modified tendon portion scaffold, and GDF-7 remains 35.17 + -2.08% on the C-GDF-7 modified tendon portion scaffold.

Referring to the above-mentioned loading methods of the growth factor and the scaffold, C-BMP-2 or BMP-2 is loaded on the bone sleeve scaffold 1, C-TGF-beta 3 or TGF-beta 3 is loaded on the cartilage sleeve scaffold 2, C-GDF-7 or GDF-7 is loaded on the tendon scaffold 3, respectively, and a C-BMP-2 modified bone sleeve scaffold, a C-TGF-beta 3 modified cartilage sleeve scaffold, and a C-GDF-7 modified tendon scaffold are constructed, respectively.

And injecting 3% of pentobarbital sodium euthanasia animals through auricular veins at the 4 th week and the 16 th week after the reconstruction of the beagle ACL injury, and collecting the in-vivo repair function of the dog femur-anterior cruciate ligament-tibia complex specimen evaluation function optimization three-phase bionic sleeve ACL graft. The specimen is stored in a refrigerator at minus 80 ℃, and then the observation of the gross morphology, the detection of Xradia imaging, the detection of histology and the detection and evaluation of biomechanics are carried out.

As shown in fig. 16, beagle specimens were collected at 4 weeks and 16 weeks after surgery, and it was found that the effect of the three-phase bionic sleeve stent (SATS) loaded with collagen binding property was better than that of autologous tendon transplantation (CTL) as seen from appearance photographs of the distal femur and proximal tibia of the knee joint, and NET in the figure refers to normal tendon interface tissue.

As shown in fig. 17, two sets of HE staining and MT staining (massson trichrome) comparisons of supra light source Collagen (Collagen distribution) and Proteoglycan (Proteoglycan distribution) were performed at 4 and 16 weeks post-surgery histological examination. H & E staining observation shows that the experimental group has obvious gradient fibrocartilage structure generation in the bone tunnel after 16 weeks of operation, while the control group is mainly made of disordered collagen fibers. The histological scoring result shows that the score of the experimental group is significantly higher than that of the control group, and the Shanghai light source Collagen (Collagen distribution) and Proteoglycan (Proteoglycan distribution) Masson staining shows that the experimental group can see that the new bone tendon interface tissue generates a large amount of chondrocytes and cartilage matrixes at 4 weeks and 16 weeks after the operation, and the new Collagen fibers and Proteoglycan are regularly arranged at 16 weeks.

As shown in fig. 18 to 20, the neonatal bone tendon interface histology score, Fibrocartilage thickness (Fibrocartilage thickness) and cartilage matrix optical density (Integrated optical density) were semi-quantitatively analyzed using image analysis software at 4 weeks and 16 weeks after surgery.

As shown in fig. 21 to 24, after 4 weeks and 16 weeks of operation, three-dimensional reconstructed images of regenerated bone and region of interest (ROI) in bone tunnel of canine anterior cruciate ligament were observed, and Xradia imaging examination showed that bone volume fraction (BV/TV), trabecular thickness (tb.th) and trabecular number (tb.n) of three-phase bionic socket scaffold (sat) were significantly increased as compared with control group (CTL).

As shown in fig. 25 and 26, the test results are 4 weeks and 16 weeks post-operation biomedical tests, and show that the tensile load (failure load) and stiffness (stiff) of the three-phase bionic sleeve stent (sat s) are significantly increased compared with those of the control group (CTL).

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

Claims

1. The utility model provides a bionical sleeve support of three-phase, a serial communication port, including bone sleeve support (1), cartilage sleeve support (2) and tendon support (3), bone sleeve support (1) with cartilage sleeve support (2) are hollow cylinder type structure, tendon support (3) are the cylinder type structure, the outside cover of tendon support (3) is equipped with cartilage sleeve support (2), cartilage sleeve support (2) overcoat is equipped with between bone sleeve support (1) and cartilage sleeve support (2), cartilage sleeve support (2) with be the tight fit between tendon support (3).

2. The three-phase biomimetic sleeve scaffold according to claim 1, wherein the bone sleeve scaffold (1) is a decellularized bone sleeve scaffold, the cartilage sleeve scaffold (2) is a decellularized cartilage sleeve scaffold, and the tendon scaffold (3) is a decellularized tendon scaffold.

3. The three-phase biomimetic sleeve scaffold according to claim 2, wherein the bone sleeve scaffold (1) is a C-BMP-2 modified decellularized bone sleeve scaffold, the cartilage sleeve scaffold (2) is a C-TGF- β 3 modified decellularized cartilage sleeve scaffold, the tendon scaffold (3) is a C-GDF-7 modified decellularized tendon scaffold, the C-BMP-2 is bone morphogenetic protein-2 with collagen binding properties, the C-TGF- β 3 is transforming growth factor- β 3 with collagen binding properties, and the C-GDF-7 is growth differentiation factor 7 with collagen binding properties.

4. A preparation method of a three-phase bionic sleeve support is characterized by comprising the following steps:

s1, preparing a bone sleeve bracket (1), a cartilage sleeve bracket (2) and a tendon bracket (3): the method comprises the following steps of trimming canine lumbar vertebra tissues into a hollow sleeve structure to obtain a bone sleeve support (1), trimming canine floating rib cartilage tissues into a hollow sleeve structure to obtain a cartilage sleeve support (2), trimming canine achilles tendon tissues into a cylindrical structure to obtain a tendon support (3), wherein the inner diameter of the bone sleeve support (1) is equal to the outer diameter of the cartilage sleeve support (2), and the inner diameter of the cartilage sleeve support (2) is equal to the diameter of the tendon support (3);

s2, carrying out cell removing treatment on the bone sleeve bracket (1), the cartilage sleeve bracket (2) and the tendon bracket (3): cleaning a bone sleeve bracket (1), a cartilage sleeve bracket (2) and a tendon bracket (3) in a phosphate buffer solution, then performing freeze-thaw cycle, rinsing with a double-resistant phosphate buffer solution, putting the rinsed bone sleeve bracket into a nuclease solution for digestion, and driving the double-resistant phosphate buffer solution to rinse by using a negative pressure suction mode to obtain a decellularized bone sleeve bracket, a decellularized cartilage sleeve bracket and a decellularized tendon bracket;

5. The method for preparing a three-phase bionic sleeve stent according to claim 4, wherein in step S3, the saturation loading capacity of the C-BMP-2 on the decellularized bone sleeve stent is 10 to 12 μmol/L, the saturation loading capacity of the C-TGF-beta 3 on the cellcartilage sleeve stent is 8 to 10 μmol/L, and the saturation loading capacity of the C-GDF-7 on the decellularized tendon sleeve stent is 10 to 12 μmol/L.

6. The method for preparing a three-phase bionic sleeve bracket according to claim 4 or 5, wherein in step S2, the phosphate buffer solution is washed for at least 30min at 3 times, and/or the double-resistant phosphate buffer solution is rinsed for at least 30min at 3 times; and/or in step S2, the temperature of the nuclease solution is 37 ℃, the nuclease solution is digested for at least 2 hours, and the nuclease solution consists of 100-200U/mL of DNase I and 80-120 mu g/mL of RNase A.

7. The method for preparing the three-phase bionic sleeve bracket according to the claim 4 or 5, wherein in the step S2, the freeze-thaw cycle comprises two steps of freezing in liquid nitrogen for 2min to 5min and thawing in a thermostatic water bath at 37 ℃ for 10min to 15min, and the number of the freeze-thaw cycle is 5.

8. The method for preparing a three-phase bionic sleeve stent according to claim 4 or 5, wherein in step S3, the C-BMP-2 is BMP-2 with collagen binding property, the C-TGF-beta 3 is TGF-beta 3 with collagen binding property, the C-GDF-7 is GDF-7 with collagen binding property, and the C-BMP-2, C-TGF-beta 3 and C-GDF-7 are purified by the biosynthesis of Detai.

9. The method for preparing a three-phase bionic sleeve stent according to claim 4 or 5, wherein in step S1, the inner diameter of the bone sleeve stent (1) is 3.8mm to 4.2mm, the outer diameter of the bone sleeve stent (1) is 4.3mm to 4.7mm, the length of the bone sleeve stent (1) is 18mm to 22mm, the inner diameter of the cartilage sleeve stent (2) is 3.3mm to 3.7mm, the outer diameter of the cartilage sleeve stent (2) is 3.8mm to 4.2mm, the length of the cartilage sleeve stent (2) is 18mm to 22mm, the diameter of the tendon stent (3) is 3.3mm to 3.7mm, and the length of the tendon stent (3) is 53mm to 57 mm.

10. The method for preparing a three-phase bionic sleeve stent according to claim 4 or 5, wherein before the step S4, the C-BMP-2 loaded decellularized bone sleeve stent, the C-TGF-beta 3 loaded decellularized cartilage sleeve stent and the C-GDF-7 loaded decellularized tendon stent obtained in the step S3 are lyophilized and sterilized, and stored at an ultra-low temperature of-60 ℃ to-80 ℃ for later use.