WO2022265585A1

WO2022265585A1 - Method of fabricating an implantable construct and an implantable construct derived from the same

Info

Publication number: WO2022265585A1
Application number: PCT/TH2021/000036
Authority: WO
Inventors: Prasit Pavasant; Daneeya CHAIKIAWKEAW; Narach KHORATTANAKULCHAI; Kaewta RATTANAPISIT; Waranyoo POOLCHAREON
Original assignee: Chulalongkorn University
Priority date: 2021-06-18
Filing date: 2021-06-18
Publication date: 2022-12-22

Abstract

A method of fabricating an implantable construct for a subject. The method comprises the step of providing a scaffold having a porous matrix, the scaffold being biocompatible to the subject; coating the scaffold with a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. I or SEQ ID No. 2; culturing cells capable of osteogenic differentiation on the coated scaffold; and acquiring the implantable construct upon attaining the osteogenic differentiation by the cultured cells.

Description

METHOD OF FABRICATING AN IMPLANTABLE CONSTRUCT AND AN IMPLANTABLE CONSTRUCT DERIVED FROM THE SAME

Technical Field

The present disclosure refers to a method for fabricating a bio-engineered construct implantable to a subject for treating injuries or deficiencies relating to bones of a subject. Particularly, the method discloses a way to produce the implantable construct by promoting osteogenic di ferentiation of one or more selected group of cells seeded on a biocompatible scaffold. The present disclosure also associates to a construct derived from the mentioned method. Also, the present disclosure includes a method for promoting osteogenic differentiation on selected cell group using one or more peptide types.

Background

Tissue engineering has been employed in years as a means for the treatment of bone related injuries and deficiencies. Bioengineered implants are generally composed of a biocompatible scaffold, a cellular coating deposited on the scaffold, and one or more reagents capable of promoting the desired properties of the cellular coating. Significant number of researches have been directed to finding materials, reagents and/or any combinations effective in assembly of a bioengineered construct best serving as an implant for the patient to treat or relieve the bone-related injuries without much side effects. For example, United States patent no. 10307514 teaches a porous scaffold formed using biodegradable microspheres that the scaffold is in a continuous gradient of pores from one end to another end. The gradient arrangement allows the bioengineered implant derived thereof being mimic to the zonal structure of natural articular cartilage. Apart from scaffold materials, effort has been put into discovering bioactive reagents with osteoconductive and/or osteoinductive characteristics to stimulate bone formation or osteo-differentiation of selected cell types on the cellular coating seeded onto the scaffold such that the bioengineered construct can be implanted free from any rejection by the host receiving the implant. United States patent no. 4394370 offers a bone graft material with osteoinductive property comprising a mixture of reconstituted collagen, solubilized bone morphogenic protein and demineralized bone particles. Furthermore, Beertsen and Van den Bos, provide another implant material comprising biocompatible carrier of a densely organized fibrous structure having fibrillar collagen in combination with a phosphatase (Apase) in an effective amount having an enzyme activity of at least 1.0 milliunit Apase per 1.0 microgram (pgm) of hydroxy proline for promoting in vivo mineralization of the material. Also, Soo et al. teaches a method of inducing bone formation of a subject in United States patent no. 10335458 in which the method includes administering a scaffold incorporated with human NELL polypeptides, bone morphogenic protein 2 and hyaluronan to the subject.

Other than the composition described above, Osteopontin (OPN) or a specific fragment of recombinant OPN has come into attention of the inventors of the present disclosure for promoting osteo differentiation in one or more selected cell types. Particularly, OPN is a major non- collagenous protein found within the bone matrix. This protein is also found in extracellular matrix in many tissues and in soluble form in body fluid. OPN participates in many biological functions including cell adhesion, migration, and biomineralization (McKee and Nanci 1996b; Sodek et al. 2000). Moreover, the expression of OPN is also found associated with bone remodeling process (Liaw et al. 1998). The soluble form of OPN has been shown to play role in inflammatory reactions while the matrix form of this protein assists in the attachment of both osteoblasts and osteoclasts to the bone surface and regulate biomineralization (Wesson et al. 2003). OPN contains at least three functional domains. The cell binding domain is located in the middle region with heparin- binding sites and calcium-binding site located in the C-terminal region (Kahles et al. 2014; O'Regan and Berman 2000). OPN has been considered as an inhibitory molecule for bone formation. OPN-<xVp3 integrin interaction could modulate intracellular Ca²⁺ pump leading to increase resorption activity of mature osteoclast (Singh et al. 2018; Tanabe et al. 2011) and stimulates osteoclast resorptive activity (Chellaiah and Hruska 2003; Ross et al. 1993). OPN also contains ASARM motif (the acidic serine-and aspartate-rich) that can bind to hydroxyapatite crystals (HA) and inhibit extracellular matrix mineralization (Iline-Vul et al. 2020). In addition, phosphorylation of OPN has been considered to involve in HA formation by enhancing OPN ability to absorb HA molecules and inhibit HA crystalize (Boskey et al. 2012; Jono et al. 2000; Wesson et al. 2003). Moreover, OPN could bind to extracellular Ca2+ via the calcium binding site (Klaning et al. 2014).

Regarding the role of OPN in bone remodeling, it has been shown that after the bone resorption, osteoclast deposit OPN at the surface of the Howship’s lacuna to regulate the recruitment and differentiation of osteoblasts (Luukkonen et al. 2019; McKee and Nanci 1996a). Interestingly, OPN null mice showed a normal development with normal bone mass and structure but increased fragility and decreased bone remodeling. These results emphasizing the function of OPN in regulating bone quality (Liaw et al. 1998; Rittling et al. 1998; Thurner et al. 2010). Still, the association between OPN and bone formation is unclear even in the presence of the above references especially for its potential application in the construction of bioengineered tissues. Thus, the present disclosure set out to offer at least a method to employ human recombinant OPN and/or a fragment of human recombinant OPN in the assembly of a bioengineered construct usable for the treatment of bone related injuries or deficiencies and an implantable construct derived from the mentioned method.

Summary

The present disclosure is directed to a method for assembling an engineered tissue construct applicable as an implant to treat or relieve bone-related injuries. Particularly, the disclosed method capitalizes on the interaction of at least a fragment of peptide originated from OPN and a selected cell group to enhance osteo differentiation of the selected cell group seeded on a carrier or scaffold.

Another object of the present disclosure is to furnish the operable optimal concentration of the mentioned peptide in promoting osteo differentiation of the selected cell group used as a cellular coating of the scaffold without adversely affecting growth of the cells.

Further object of the present disclosure pertains to a tissue-engineered or bioengineered construct obtained through the aforesaid method. The construct is implantable to a subject, preferably a human subject, to at least alleviate a diseased state associated to injuries or deficiencies of bone and the like. The disclosed construct can be implanted to the subject without substantially facing any rejection from the host body immune system.

Still, another object of the present disclosure regards to a method of promoting osteo differentiation in a selected cell group by way of administering a human recombinant OPN and/or a predetermined fragment of the human recombinant OPN.

At least one of the preceding objects is met, in whole or in part, by the present disclosure, in which one of the embodiments of the present disclosure is a method of fabricating an implantable construct for a subject comprising providing a scaffold having a porous matrix with the scaffold being biocompatible to the subject; coating the scaffold with a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2; culturing cells capable of osteogenic differentiation on the coated scaffold; and acquiring the implantable construct upon attaining the osteogenic differentiation by the cultured cells. For a number of embodiments of the disclosed method, the peptide of amino acid sequence of SEQ ID No. 1 coated on the scaffold has a concentration ranged between 5 - 20 ng/cm²

Furthermore, the peptide of amino acid sequence of SEQ ID No. 2 coated on the scaffold has a concentration preferably not exceeding 15 ng/cm²

For more embodiments, the cells are selected from any one or combination of periodontal ligament stem cells, and bone marrow stromal cells.

According to some embodiments, the osteogenic differentiation of the disclosed method is by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cultured cells.

For several embodiments, the peptide is recombinant peptide produced from N. benthamiana.

Another aspect of the present disclosure refers to a bioengineered construct implantable to a subject comprising a scaffold having a porous matrix that the scaffold is biocompatible to the subject; a coating being deposited to the scaffold, the coating comprising a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2; and cells capable of osteogenic differentiation being seeded and cultured on the scaffold substantially contacting with the coating. Preferably, the cells become osteogenic differentiated by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cultured cells.

According to some embodiments of the disclosed construct, the peptide of amino acid sequence of SEQ ID No. 1 coated on the scaffold has a concentration ranged between 5 - 20 ng/cm².

In accordance with several embodiments of the mentioned construct, the cells are selected from any one or combination of periodontal ligament stem cells, and bone marrow stromal cells. More preferably, the cells are of autologous origin.

More aspect of the present disclosure encompasses the use of a peptide having amino acid sequence as setting forth in SEQ ID No. I or SEQ ID No. 2 for initiating osteogenic differentiation of a cell selected from a group consisting of periodontal ligament stem cells, and bone marrow stromal cells by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cell.

Still, an aspect of the present disclosure relates to a method of promoting osteo differentiation in periodontal ligament stem cells comprising the steps of brining recombinant human OPN peptide of SEQ ID no. 2 and/or a fragment of recombinant human OPN peptide of SEQ ID no. 1 to periodontal ligament stem cells cultured on a platform in the presence of a medium. Preferably, the amount of the peptide is in a range of concentration, in relation to the surface area of the platform, at 5 - 20 ng/cm² to sufficiently yield the desired results.

Brief Description of Drawings

Fig. 1 is a flowchart showing one embodiment of the present disclosure in producing the bioengineered construct;

Fig. 2 shows various test results about PDLSCs seeded on OPN-coated plates with a density of 150,000 cells/well in 12-well plates for 24 hours with the plates being coated with different concentrations of OPN ranging from 0, 7.5, 15 and 30 ng/cm2, where (A) is RT-PCR analysis revealed that coated FL-OPN induced the expression of osterix (OSX) in a dose-dependent manner with the expression of OSX being determined by real-time RT-PCR and normalized to the expression of GAPDH, (B) is a gel picture of Western Blot showing increased expression of osterix protein by PDLSCs seeded on the OPN-coated plate for 24 hours and actin was used as loading control, (C) is another RT-PCR analysis on OSX expression in the cultured cells with soluble FL-OPN added, (D) are pictures of plates culturing the PDLSCs with plant produced OPN and commercial OPN produced from HEK-263 cells being used at two different concentration of 15 and 30 ng/cm² respectively in osteogenic medium (OM) for 14 days while cells cultured on uncoated surface of growth medium (GM) and OM were used as negative and positive controls, (E) is a RT-PCR analysis results with regard to abolished OPN- induced expression of osterix in the addition of K02288 - the ALK- 1 inhibitor - in the cell culture, and (F) is gel pictures of Western blot analysis supporting the finding indicated in (E) (* and ** indicate significant differences from control, p>0.05, 0.01, respectively);

Fig. 3 includes different diagrams about molecular docking of extracellular domain of ALK 1 and calcium binding site of OPN where (A) is a schematic representation showing the three function domains of the full length osteopontin (FL-OPN) (modified form Wai and Kuo 2008) and the three constructs generated therefrom including N142 (the N-terminal part of OPN contained Integrin binding domain), C-I22 (the C-terminal part of OPN contain Calcium binding domain but not heparin binding domain) and Cl 226 (the C122-OPN that 1 1 amino acid in the middle of calcium bind domain was deleted illustrated in Fig. 6), (B) and (C) respectively illustrate the predicted peptide structure of calcium binding site of OPN and the extracellular domain of ALK 1 , (D) shows the in silico binding of calcium binding site of OPN and the extracellular domain of ALK1, and (E) further illustrates the Interactions between calcium binding site and extracellular domain of ALK1;

Fig. 4 (A) is RT-PCR analysis and (B) is Western blot analysis about PDLSCs seeded on Cl 22- coated surface, (C) is another RT-PCR analysis relating to expression of OSX in PDLSCs seeded on N 142- and CI226-coated surface, (D) includes picture of cells culture plates showing in vitro calcification of the cells examined through Alizarin Red staining on C122-coated surface, (E) is a

RT-PCR analysis showing K02288 of 2 nM inhibiting the inductive effect of C 122 ( 15 ng/cm^) on OSX expression, and (F) shows gel pictures respectively indicating that capability of Cl 22 for increasing the level of p-Smad-1 in PDLSCs after being seeded on CI22-coated surface for 6 hours and the capability being inhibited by ALK-1 inhibitor (** and *** indicate significant differences from control, p > 0.01, 0.001 , respectively. ## and ### indicate significant differences from Cl 22 7.5 and 15 ng/cm2, p > 0.05, 0.01, respectively);

Fig. 5 showing results about two circular defect with 5-mm in diameter created in the calvaria of Wistar rats and subsequently treated the defects by inserting the uncoated scaffold (Sc) or Cl 22- coated scaffolds (C 122-Sc) into the defect and kept for 4 weeks (5A and 5B) and 8 weeks (5C and 5 D) to monitor amount of new bone formation at the treated site using DCT analysis, where Fig. 5 A and 5C respectively show the defect on the left hand side of the calvariae using Cl 22-Sc and on the right hand side using the sham-operated control defects, Fig. 5B and 5D respectively show the defect on the left hand side of the calvariae using Cl 22-Sc and on the right hand side using uncoated Sc, Fig. 5E is a graph presenting the computed amount of new bone formation revealing that Cl 22-Sc had significantly enhanced the new bone formation compared to Sc and sham experiment (p > 0.05), and Fig. 5F shows histological slides of calvaria stained with Masson’s Trichrome after the treatment indicating that the fibrous tissue established in sham defect while new bone formed in defects filled with both Sc and C122-Sc (*, *** indicated the significant compared to Sham at p > 0.05 and 0.001 , respectively);

Fig. 6 shows amino acid sequence alignment of full length OPN (FL-OPN or SEQ ID No. 2) with regard to the three constructs C-122-OPN (or SEQ ID No. 1), C1226-OPN (or SEQ ID No. 11) and N- 142 (or SEQ ID No. 12) generated for the analysis performed in the present disclosure with C1226-OPN having a I I amino acid deleted region (DWDSRGKDSYETS) compared to the counterpart, C 122-OPN; and

Fig. 7 shows amino acid sequences of SEQ ID No. I and SEQ ID No. 2.

Detailed Description

The present disclosure may be embodied in other specific forms without departing from its structures, methods, or other essential characteristics as broadly described herein and claimed hereinafter. The described embodiments are to be considered in all respects only as illustrative, and not restrictive. The scope of the disclosure is, therefore, indicated by the appended claims, rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

The term “polypeptides” used herein throughout the disclosure refers to a chain of amino acids linked together by peptide bonds but with a lower molecular weight than protein. Polypeptides can be obtained by synthesis or hydrolysis of proteins. Few polypeptides can be joined together by any known method in the art to form a functional unit.

As used herein, the terms “approximately” or "about", in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.

According to one aspect of the present disclosure, a method of fabricating an implantable construct for a subject is described hereinafter. The disclosed method essentially comprises providing a scaffold having a porous matrix, the scaffold being biocompatible to the subject; coating the scaffold with a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2; culturing cells capable of osteogenic differentiation on the coated scaffold; and acquiring the implantable construct upon attaining the osteogenic differentiation by the cultured cells.

Pursuant to the embodiments of the disclosed method, the scaffold or carrier is preferably fabricated from a material, polymer, copolymer, composite or alloy which is substantially biocompatible or immunologically inert with respect to the immune system of the subject, host or recipient receiving the bioengineered construct with no, almost no or minimal effect in triggering any immunological reactions in the subject potentially leading to rejection of the implanted construct. More preferably, the material used for fabrication of the construct is biodegradable over a period of time upon implanting to the host. The construct of the scaffold may eventually be absorbed or resorbed by the body of the host. The scaffold or carrier can be made of polymer or copolymer Polycaprolactone (PCL), Polyethylene Glycol (PEG), Poly(glycolic acid), PoIy(lactic acid), polyurethanes or any derivatives acquired thereof. Copolymer of PCL-PEG or triblock of PEG-PCL-PEG is generally employed in the more preferred embodiments. For more embodiments, the scaffold may be shaped and dimensioned to substantially conform the part of the deficiencies or injuries to be treated such that the construct can be placed thereto to promote formation of bone and/or other connective tissues such as collagen, fibrous, cartilage, etc. required to at least alleviate the diseased state caused by the deficiencies and/or injuries. Furthermore, the scaffold may be made porous to promote adhesion and growth of the cells seeded thereto. The porous matrix or nature of the scaffold enables better coating and adherence of the cells or differentiated cells seeded thus shielding the scaffold entirely from initiating any undesired immunological reaction in the host upon implantation of the construct. It is worth to note that the pore size applied to the scaffold can be varied according to the cell type used and the deficiencies to be treated. Preferably, the pore size may range from 100 to 500 microns.

Referring to Fig. 7, the amino acid sequences of the peptides employed for coating on the surface of the scaffold are illustrated. Specifically, the peptide of SEQ ID No. 1 is a truncated fragment derive from the FL-OPN. The peptide of SEQ ID No. 1 is specially designed by the inventors of the present disclosure to include only the calcium-binding domain, where function of which is uninterrupted for starting one or more cellular cascade reactions including osteo differentiation to assist assembly of the mentioned construct. On the other hand, the peptide of SEQ ID No. 2 refers to the FL-OPN carrying the calcium-binding domain as well. For a number of embodiments, the peptide of SEQ ID No. 2 is a recombinant peptide produced from or expressed by N. benthamiana. The genetic sequence encoding for the peptide of SEQ ID No. 2 may have been modified to best suit expression system present in N. benthamiana. The present disclosure, in some experiments performed (not shown), found that human recombinant OPN (rhOPN) generated by N. benthamiana possesses post-translational modifications similar to the process found in mammalian cells. The plant-yielded rhOPN used in the present disclosure has a molecular weight about 60 kDa, which is similar or almost similar to the HEK-293 produced rhOPN available commercially. Moreover, plant-yielded rhOPN also presents a secondary and tertiary structure equivalent to commercially available rhOPN. Alternatively, both peptides of SEQ ID No. 1 and SEQ ID No. 2 can be synthesized chemically to be employed for the disclosed method.

For more embodiments, the peptides coated on the construct preferably mimic or take the form of matrix-bound such that the utilized peptides facilitate osteo differentiation of the cells seeded on the construct and brought into contact with peptides. Particularly, the inventors of the present disclosure discovered that the soluble form of the aforesaid peptides fail to initiate osteo- differentiation of the seeded cells in the experiments performed. The failure of soluble peptides in triggering the desired osteo-differentiation in the selected cell group may be caused by deformed peptide folding of the soluble form rendering malfunctioning of the calcium-binding resided within the mentioned peptides. Therefore, the matrix-bound or non-soluble form of the peptides of the SEQ ID No. I or SEQ ID No. 2 are utilized for establishing the mentioned bioengineered construct. To render the construct incorporated with the non-soluble or matrix-bound construct, a double leaching approach is preferably employed. For example, the scaffold sized to the preferred dimension is treated with Sodium Hydroxide solution to create hydrophilic surface on the construct followed by washing the treated scaffold thoroughly in deionized distilled water, vacuum-drying and sterilization. The peptides of predetermined concentration are then added onto the sterilized scaffold then incubating scaffold at room temperature to coat the peptides onto the surface of the scaffold. The coated scaffold can be stored at a temperature of 4°c or below until use.

In accordance with some embodiments, the peptide of amino acid sequence of SEQ ID No. 1 coated on the scaffold has a concentration ranged between 5 - 20 ng/cnr. Alternatively, the peptide of amino acid sequence of SEQ ID No. 2 coated on the scaffold has a concentration not exceeding 15 ng/cm². Through the experiments performed and described hereinafter, the present disclosure revealed that the rate of the osteo-differentiation promoted by the mentioned peptides on the construct only meets the first order reaction or positively correlates to the concentration of the peptides available on the construct within a given concentration range of the peptides. Once the concentration of the peptides available on the construct exceeds the given range, the osteo- differentiation effect becomes plateau. More interestingly, the highly concentrated peptides may assert an inhibitory effect against osteo-differentiation due to over aggregation of the peptides on the construct. The unwanted aggregation may further lead to shielding of the calcium binding domains on the peptides and blocking the calcium binding domain from interacting with the corresponding receptor on the cells seeded to the construct for beginning one or more cellular reactions triggering the osteo-differentiation.

Referring to setting forth description, the scaffold is further deposited with cells capable of osteogenic differentiation on the surface coated with the mentioned peptides. It is important to note that there are embodiments of the disclosed method utilizing more than one cell types for seeding and culturing on the scaffold coated with the peptides to achieve the desired therapeutic outcome on the subject receiving the construct. Among the multiple cell types seeded in these embodiments, at least one of the cell types is capable of reacting with the coated peptide to become osteo-differentiated. Preferably, the cells capable of becoming osteo-differentiated is selected from any one or combination of periodontal ligament stem cells and bone marrow stromal cells. The amount or concentration of the cells seeded and/or cultured on the scaffold preferably ranges from 0.1 - 5.0 ^c 10⁵ cells/cm². Furthermore, in some embodiments, the culturing step is conducted in the presence of a culturing medium. The medium can be osteo-inductive to enhance the rate of osteo-differentiation on the scaffold. By culturing the cells in the presence of a suitable medium, the tissue engineered construct can be prepared faster for subsequent procedure to be implanted to the subject. In some embodiments, the construct becomes implantable when the cultured cells have reached about 50% to 100% osteo-differentiation.

As demonstrated in the experiments described hereinafter, the osteo-differentiation is activated through interaction of the calcium binding resided in the coated peptides and the ALK-I receptor located on the surface of culturing or cultured cells. Under given circumstances, such interaction may be required to continue even after the construct was implanted to the subject. The osteo- differentiation cells seeded on the scaffold will continue to grow and potentially further differentiate to attain the desire therapeutic outcome at the site which the implant was placed. Therefore, it is crucial that the subject is free of any medication comprising inhibitor of ALK-I receptor or manufacturer based upon known clinical ALK-I inhibitor such as PF-3446962, RAP- 041, etc. The subject may preferably be cleansed of any ALK-1 inhibitor drugs before subjecting to the procedures putting the tissue engineered construct into the subject.

Further aspect of the present disclosure pertains to a bioengineered or tissue-engineered construct implantable to a subject. The disclosed construct can be acquired through the aforesaid method with or without further modification. Particularly, the construct comprises a scaffold having a porous matrix that the scaffold is preferably biocompatible to the subject; a coating being deposited to the scaffold with the coating comprising a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2; and cells capable of osteogenic differentiation being seeded and cultured on the scaffold substantially contacting with the coating.

Accordingly, the cells deposited onto the scaffold become osteogenic differentiated by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cultured cells.

As described in the setting forth, the scaffold or carrier is preferably fabricated from a material, polymer, copolymer, composite or alloy which is substantially biocompatible or immunologically inert with respect to the immune system of the subject to avoid or at least minimize any immunological reactions potentially initiated leading to rejection of the implanted construct. The scaffold or carrier of the disclosed construct can be made of polymer or copolymer of Polycaprolactone (PCL), Polyethylene Glycol (PEG), Poly(glycolic acid), Poly(lactic acid), polyurethanes or any derivatives acquired thereof. Copolymer of PCL-PEG or triblock of PEG- PCL-PEG is generally employed in the more preferred embodiments. These polymer or copolymer possesses the biodegradable property allowing the implanted construct to be resorb by the body of the subject or recipient. Likewise, the scaffold may be shaped and dimensioned to substantially conform the part of the deficiencies or injuries to be treated such that the construct can be placed thereto to promote formation of bone and/or other connective tissues such as collagen, fibrous, cartilage, etc. required to at least alleviate the diseased state caused by the deficiencies and/or injuries. Furthermore, the scaffold may be made porous to promote adhesion and growth of the cells seeded thereto. The pore size applied to the scaffold can be varied according to the cell type used and the deficiencies to be treated. Preferably, the pore size may range from 100 to 500 microns.

For a number of embodiments of the disclosed construct, the peptide of amino acid sequence of SEQ ID No. 1 coated on the scaffold has a concentration ranged between 5 — 20 ng/cm². Alternatively, the peptide of amino acid sequence of SEQ ID No. 2 coated on the scaffold has a concentration not exceeding 15 ng/cm². Like being demonstrated in the examples furnished herein, the rate of the osteo-differentiation promoted by the mentioned peptides on the construct only meets the first order reaction or positively correlates to the concentration of the peptides available on the construct within a given concentration range. Once the concentration of the peptides available on the construct exceeds the given range, the osteo-differentiation effect becomes plateau. Also, the inventors of the present disclosure observed that the highly concentrated peptides may assert an inhibitory effect against osteo-differentiation due to over aggregation of the peptides on the construct. Moreover, the peptides used in the disclosed construct is preferably of non-soluble or matrix-bound form. The present disclosure found that the soluble form of the aforesaid peptides fails to initiate osteo-differentiation of the seeded cells in the experiments performed. Such failure may be caused by deformation of the soluble fold peptides compared to the matrix-bound counterparts.

Referring to some embodiments of the disclosed construct, more than one cell types can be deposited or positioned on the scaffold preferably covering the entire or almost entire external surface of the scaffold to reduce the likelihood of resulting the host rejection in the presence of the immunologically foreign scaffold despite biocompatible and/or biodegradability of the scaffold. Preferably, the cells capable of becoming osteo-differentiated is selected from any one or combination of periodontal ligament stem cells, and bone marrow stromal cells. The amount or concentration of the osteo-differentiated cells on the scaffold preferably ranges from 0.1 - 5.0 * IO⁵ cells/cm². In some embodiments, the construct becomes implantable when the cultured cells have reached about 50% to 100% osteo-differentiation. According to several embodiments, the cells seeded and cultured on the scaffold are harvested from the subject directly and being applied for the assembly of the disclosed construct. Specifically, the cells cultured on the scaffold is of autologous origin to minimize any immunological rejection of the implanted construct.

Further aspect of the present disclosure relates to the use of a peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2 for initiating osteogenic differentiation of a cell selected from a group consisting of periodontal ligament stem cells, and bone marrow stromal cells by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cell.

In accordance with another aspect, the present disclosure refers to a method of promoting osteo differentiation in periodontal ligament stem cells comprising the steps of bringing recombinant human OPN peptide of SEQ ID no. 2 and/or a fragment of recombinant human OPN peptide of SEQ ID no. I to periodontal ligament stem cells cultured on a platform in the presence of a medium. Preferably, the amount of the peptide is in a range of concentration, in relation to the surface area of the platform, at 5 - 20 ng/cnr to sufficiently yield the desired results.

The following example is intended to further illustrate the disclosure, without any intent for the disclosure to be limited to the specific embodiments described therein.

Example 1

PDLSCs were prepared as previously described (Rattanapisit et al. 2017). A written informed consent was given by each donor and was approved by the human ethical committee, Faculty of Dentistry, Chulalongkom University (HREC-DCU 2018-054). These experiments of the present disclosure were conducted in accordance with the Helsinki Declaration. Human PDLSCs were isolated from periodontal ligament scraped from the middle-third of the root. The explants were cultured with a high glucose-Dulbecco modified eagle medium (DMEM) containing 10% fetal bovine serum, 2 mM of L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml) and amphotericin B (5 mg/ml) (all reagents from Gibco, Sigma, St. Louis, Missouri, USA.) and maintained in a humidified atmosphere of 5% CO2 at 37 °C. After confluency, PDLSCs were detached and sub-cultured at a ratio of 1 :3. Cells from the third to the fifth passages were used in the experiments.

For the inhibition experiments, cells were pretreated for 30 min with 2 nM of the specific ALK-1 inhibitor, K02288 (Sigma, St. Louis, Missouri, USA.) before their seeding on the OPN coated surface.

Example 2

Full-length OPN (FL-OPN) gene was optimized to conform N. bentamiana codon and synthesized as previously described (Rattanapisit et al. 2017). The protein contains a signal peptide (SP) at N- terminus and an 8-His tag at C-terminus. The N-terminus half contains 142 amino acids (N142) and the C-terminus without heparin binding domain (C 122) were generated and amplified by using pairs of primers: SEQ ID No. 3 and SEQ ID No. 4, and SEQ ID No. 5 and SEQ ID No. 6, respectively. The calcium binding domain within C 122 was modified by site-directed mutagenesis (WDSRGKDSYET) The modified C 122 or C 1225 was generated by using pairs of primers; SP-F SEQ ID No. 7 and SEQ ID No. 8, and SEQ ID No. 9 and SEQ ID No. 10. The list of primers was shown in the Table 1 as follows.

Table 1

Example 3

Cell culture plates (Thermo scientific) were coated with rhOPN to obtain the final concentration of 7.5, 15 and 30 ng/cm2. Coating was performed on a shaker overnight at 4 °C and protected from light. The coated surface was air dried before seeding cells. Cells were seeded at the density of 4x 104 cell/cm2. For examining the effect of soluble OPN, confluent cells were treated with 50, 100, 200 ng/ml of FL-OPN. Total RNA was extracted, reversed transcribed and subjected to real time PCR analysis. Quantitation of expression was calculated based on the quantitation cycle (Cq) following normalization to expression of glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH). Primer sequence was shown in supplement Table 1. The PDLSCs lysate were extracted using radio immunoprecipitation (R1PA) buffer (50 mM Tris/HCL, 150 mM NaCI, 1 mM EDTA, 1% Nonidet P-40, 0.25% Na-deoxycholate) supplemented with proteases inhibitor cocktail (Sigma). The total protein concentration was measured by BCA protein assay kit (Pierce Biotechnology, Rockford, IL). An equal amount of protein from each sample was loaded into 12% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis, transferred to nitrocellulose membrane and incubated with antibody against human OPN, OSX, Smad- 1 ,5,8 and p-Smad- 1 ,5,8 followed by incubating with peroxidase-labeled anti-rabbit polyclonal antibody (Cell signaling). The signal was activated by chemiluminescence and captured using an image analyzer (GE). All established PDLSCs were analyzed. PDLSCs were cultured on plant produced full length Osteopontin (FL-OPN) for 24 hours. RT-PCR analysis showed that cell cultured on FL-OPN- coated surface increased the expression Osterix (OSX), a key transcriptional regulator of osteogenic differentiation, in both mRNA and protein levels (Fig.2A and 2B). The inductive effect of FL-OPN could be found in a dose dependent manner with the optimum level was at 15 pg/cnr. Interestingly, addition of soluble OPN did not have any effect on the expression of OSX (Fig.2C). Fig.2D shows that 15 pg/cnrr of both plant and HEK 293 produced OPN could enhance the in vitro calcification as judged by Alizarin red S staining as compared to the cells in osteogenic medium (OM). The 15 pg/cm² dose has been used throughout the experiments conducted since higher concentration of OPN was either no effect or inhibit the OSX expression.

Fig. 2D and 2E show the effect of ALK-1 inhibitor, K.02288. This inhibitor has been shown to bind directly to ALK-I and inhibit BMP-9-ALK1 signaling (Kerr et al. 2015). Addition of K02288 inhibited the FL-OPN-induced the expression of OSX in both mRNA and protein levels without any effect on cell viability.

Example 4

The peptide structure of the calcium binding domain (WDSRGKDSYET) of OPN was predicted by PEP-FOLD3 program (http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) (Alland et al. 2005; Neron et al. 2009). The 3D structure of the extracellular domain of ALK1 at the residue 30-104 (PDB code is 4FAO) (Townson et al. 2012) was used. The molecular docking software was iGEMDOCK (Yang and Chen 2004). The receptor was ALK1 and the ligand was calcium binding domain of OPN. All of the figures were illustrated by Discovery Studio2019 (Biovia, San Diego, CA).

To confirm the interaction between ALK-I receptor and functional domains of OPN, a molecular docking approach was used. The structure of three functional domains of OPN; integrin binding, calcium binding and heparin binding domains as well as the extracellular domain of ALK-1 were predicted using a computer program (Fig 3A). The sequence of the three functional domains of OPN was derived from the report of Wai and Kuo (Wai and Kuo 2008). Ten models of each domain were generated from the input sequences. The lowest free energy model was selected for further experiments. Fig. 3B and 3C shows the three-dimensional structure of the calcium binding domain and ALK-1, respectively. From the predicted model, the overall structure of calcium binding domain is a random coil that could interact with the extracellular domain of ALK-1 as shown in Fig. 3D and 3E. The calcium binding site of OPN bind to the extracellular domain of AL 1 with a free energy at -138.6 kcal/mol. Most of the interactions were hydrogen bonds. The interactions of amino acids of the calcium binding domain and ALK-1 were shown in Table 2

Table 2

Example 5

Double leached PCL-PEG scaffolds were fabricated as previously reported (Thadavirul et al. 2014). The circular scaffolds (5 mm diameter, 1 mm thickness) were prepared and treated with 1M Sodium Hydroxide solution (Ajax Finechem, Australia) at 37“c for I hour to create the hydrophilic surface. The treated scaffolds were washed thoroughly in deionized distilled water, vacuum-dried and sterilized by dipping in 70% v/v ethanol for 30 minutes. The 100 ml solution containing SO ng of C 122 was added into each scaffold. The scaffolds were incubated in shaking incubator for 16-18 hours at room temperature and then stored in a 4°c until used.

The experiment was carried out with 12 six-week-old Wistar rats. The protocol was approved by the Chulalongkom University Animal Care and Use Committee, Animal Use (Protocol No. 1773019). Two circular calvaria defects (5 mm in diameter) were created under general anesthesia with xylazine and ketamine intraperitoneal injection. Rats were divided into two groups with 6 rats in each group. In group I the right defect was filled with a C 122-coated scaffold and the other was left empty (sham). In group 2 the right defect was filled with scaffold without Cl 22 and the other was filled with CI22-coated scaffold. The wound was closed with a 4-0 nylon suture and euthanized at 4 and 8 weeks.

Bone formation in the calvaria defects were analyzed using mCT imaging. The samples were fixed immediately with 10% (v/v) formaldehyde for 24 hours, followed by an extensive washing with PBS. All specimens were scanned under 70 kVP, 114 mA, 8 W of X-ray. Total bone volume was analyzed based on hydroxyapatite (HA) at 1200 mg HA/cc using mCT 35 SCANCO MEDICAL and mCT evaluation program (SCANCO Medical AG, Switzerland).

The specimens were decalcified using Surgipath Decalcifier II (Leica Biosystems Richmond Inc, Richmond, IL) and processed for paraffin embedding. Sections of 5 mm thickness were cut and stained with Masson's Trichrome. Digital images were obtained using a microscope (Carl Zeiss, Germany). Cells were fixed with cold methanol for 10 minutes and washed with deionized water. The wells were stained with 1% Alizarin Red S solution (Sigma, MO) for 5 min at room temperature. The staining was analyzed by microscopy.

The data was presented as mean ± SD, statistical analyses were performed by using nonparametric t tests, one-way and two-way ANOVA followed by Tukey’s multiple comparison test. Values of P < 0.05 were considered significant. The analyses were performed using GraphPad Prism9 Software

To confirm the results from molecular docking, the present disclosure generated three OPN fragments; N142, Cl 22 and C122d that contain an integrin binding domain, calcium binding domain (CaBD) and mutated calcium binding domain, respectively. (Fig 3A). For functional analysis of C 122, PDLSCs were seeded on the C 122 coated surface and the expression of Osterix was determined by both RT-PCR and Western blot analysis. Fig. 4 shows the inductive effect of C 122 on the expression of OSX mRNA (Fig.4A) and protein (Fig.4B) in a dose dependent manner. On the contrary, cells seeded on N 142 and Cl 226 could not induced OSX expression. The optimal concentration of Cl 22 on OSX induction was found to be around 7.5-15 pg/cm². These two concentrations of C 122 could also enhance the in vitro calcification when cultured in OM medium as compared to those in OM alone (Fig.4C). Moreover, increased the level of p-Smadl could be observed in cells seeded on Cl 22 coated surface for 6 hours. Addition of K02288 could inhibit the inductive effect of Cl 22 on OSX expression and the increase level of p-Smad-1 (Fig.4D-4E). Further, the osteoinductive effect of C 122 was confirmed in the experiments conducted on a rat calvaria defect model. C-122 was coated on 3D-porous PCL scaffold and implanted in calvaria defects of Wistar rats. Fig.4A-B showed the results from mCT analysis after 4 and 8 weeks. The 3D porous scaffold supported new bone formation when compared to the sham operated defect. The amount of new bone was significantly increased when C122-coated scaffold was used. Histomorphometric analysis further supported the result from uCT analysis (Fig.5C). Sections of calvariae stained with Masson’s Trichrome showed an increase of new bone formation within the scaffold at both 4 and 8 weeks.

Example 6

Through the experiments described above, the present disclosure demonstrated the osteogenic inductive ability of FL-OPN. The ability is possibly occurred via the interaction between CaBD of OPN and ALK-1 receptor. This statement was supported by the results from specific inhibitor against ALK-1, the molecular docking model, and the ability of OPN-construct, Cl 22, which contain CaBD to increase the expression of Osx and p-Smad-1. Deletion of CaBD (Cl 226) could not increase OSX and p-Smad-1. Finally, the ability of C122-coated scaffold to support the new bone formation in the rat calvarial model further supported the function of C 122 in bone formation.

As mention earlier, the structure of OPN contain at least three functional domains (Wai and Kuo 2008). The integrin binding domain binds to anb3 integrin to promote the attachment of osteoclast and osteoblast (Denhardt and Noda 1998; Giachelli and Steitz 2000) and modulate osteoclast resorption activity. (Chellaiah and Hruska 2003; Ross et al. 1993) The heparin binding domain was proposed to regulate wound healing and inflammation via the binding to heparin-like glycosaminoglycans (Kon et al. 2008; Taylor and Gallo 2006). The CaBD, without the direct interaction with any cell surface receptor, interacted with extracellular calcium and inhibit mineralization. (Klaning et al. 2014) The results from the experiments conducted for the present disclosure demonstrated the possibility that CaBD of OPN might be able to interact with ALK-1, the BMP-9 receptor and promote osteogenic differentiation by PDLSCs. The OPN-ALK-1 interaction might restrict to the concentration and localization of OPN. High concentration of FL- OPN and soluble FL-OPN may, in fact, fail to induce OSX expression or react with the ALK-1 receptor as being observed from the experiment conducted in the present disclosure. The matrix form and soluble form of OPN or specific fragments of OPN may somehow possess different bioreactivity towards various cells and receptors thus playing wider role in regulating cellular functions. Particularly, soluble OPN involves directly in inflammatory process (Ashkar et al. 2000; iida et al. 2017) and progress of cardiovascular disease (Abdalrhim et al. 2016; Fitzpatrick et al. 1994), while matrix form effectuates about cell adhesion, biomineralization and osteogenic differentiation as illustrated by the experiments of the present disclosure.

Previously, the role of matrix OPN in osteogenic differentiation has been shown to require the interaction with collagen substrate both in vitro and in vivo (Carvalho et al. 2019; Zurick et al. 2013). However, the cell-OPN interaction that regulate bone formation was still unclear in view of the earlier research done. In the experiments of the present disclosure, the ability of FL-OPN in supporting osteogenic differentiation was found to be correlating with the relative concentration of the FL-OPN within a defined space up to certain level. Specifically, inventors of the present disclosure found that high concentration of OPN coated on the scaffold or culture plates failed to exhibit the desired osteoinductive ability. Such findings in this regard appear to be in line with some of the contradictory observations reported by other researchers about inhibitory effect of OPN on bone formation upon using soluble OPN or high concentration of matrix form OPN (Singh et al. 2018; Tanabe et al. 2011). The possible discrepancies in protein folding of the soluble and matrix form OPN may attribute to the inconsistent outcomes observed. Still, low and high concentration of matrix OPN might have different aggregation or different folding thus variably affecting the exposure of a particular functional domain resided within the OPN. It is worth noting that the amino acid sequence, pH and concentration of the OPN available for contacting or potentially interaction with the PDLSCs may assert significant influence over the folding and selfaggregation of protein, (van der Linden and Venema 2007). Huang et al., has proposed that concentration of a protein may affect the surface tension of the molecule and alter the folding of the protein (Huang et al. 2001). Therefore, it is possible that the availability of CaBD or unknown domain in Cl 22 should be taken in account for the function of OPN.

Taken together, it is possible that the osteogenic inductive ability of OPN is depended on the availability of CaBD domain to interact with ALK-1 on cell surface and this availability might depended on protein concentration or specific interaction of OPN and other ECM within the matrix. In addition, the Cl 22, OPN construct that contain CaBD could induce the expression of OSX and increase the level of p-Smadl, the signaling target of ALK-1. Deletion of CaBD in C1226 could not induce p-Smad-1 Moreover, this activation was inhibited by ALK-1 inhibitor. These results prove that there are possible interactions between the CaBD in C122 and ALK-1 leading to cascade of cellular activities promoting osteo differentiation. Also, results acquired from In vivo experiments of the present disclosure corroborate the role of Cl 22 in resulting bone formation. In conclusion, the present disclosure demonstrated that rhOPN or specific fragment of rhOPN can induce osteogenic differentiation of PDLSCs provided that the rhOPN or fragments derived thereof are prepared in the required form and/or concentration. The inductive ability of OPN is possibly occurred via the interaction between CaBD of OPN and ALK-1 receptor. The results not only provide the new concept on OPN function but also provide the new approach for bone tissue engineering application.

It is to be understood that the present disclosure may be embodied in other specific forms and is not limited to the sole embodiment described above. However, modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto.

References

Abdalrhim AD, Marroush TS, Austin EE, Gersh BJ, Solak N, Rizvi SA, Bailey KR, Kullo IJ.2016. Plasma osteopontin levels and adverse cardiovascular outcomes in the peace trial. PLoS One. I l(6):e0156965.

Alland C, Moreews F, Boens D, Carpentier M, Chiusa S, Lonquety M, Renault N, Wong Y, Cantalloube H, Chomilier J et al. 2005. Rpbs: A web resource for structural bioinformatics. Nucleic Acids Res. 33(Web Server issue):W44-49.

Ashkar S, Weber GF, Panoutsakopoulou V, Sanchirico ME, Jansson M, Zawaideh S, Rittling SR, Denhardt DT, Glimcher MJ, Cantor H. 2000. Eta-1 (osteopontin): An early component of type-1 (cell-mediated) immunity. Science. 287(5454):860-864.

Boskey AL, Christensen B, Taleb H, Sorensen ES. 2012. Post-translational modification of osteopontin: Effects on in vitro hydroxyapatite formation and growth. Biochem Biophys Res Co mun. 4l9(2):333-338.

Carvalho MS, Cabral JM, da Silva CL, Vashishth D. 2019. Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation, osteogenic differentiation, and angiogenic properties. J Cell Biochem. 120(4):6555-6569.

Chellaiah MA, Hruska KA. 2003. The integrin alpha(v)beta(3) and cd44 regulate the actions of osteopontin on osteoclast motility. Calcif Tissue Int. 72(3): 197-205.

Denhardt DT, Noda M. 1998. Osteopontin expression and function: Role in bone remodeling. J Cell Biochem. 72 Suppl 30-31 (S30-31 ):92- 102.

Fitzpatrick LA, Severson A, Edwards WD, Ingram RT. 1994. Diffuse calcification in human coronary arteries. Association of osteopontin with atherosclerosis. J Clin Invest. 94(4): 1597-1604. Giachelli CM, Steitz S. 2000. Osteopontin: A versatile regulator of inflammation and biomineralization. Matrix Biol. 19(7):615-622.

Huang W, Bhullar RS, Fung YC. 2001. The surface-tension-driven flow of blood from a droplet into a capillary tube. J Biomech Eng. 123(5):446-454.

Iida T, Wagatsuma K, Hirayama D, Nakase H.2017. Is osteopontin a friend or foe of cell apoptosis in inflammatory gastrointestinal and liver diseases? Int J Mol Sci. 19(1).

Iline-Vul T, Nanda R, Mateos B, Hazan S, Matlahov I, Perelshtein I, Keinan-Adamsky K, Althoff- Ospelt G, Konrat R, Goobes G. 2020. Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites. Sci Rep. 10( 1): 15722. Jono S, Peinado C, Giachelli CM. 2000. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem. 275(26):20197-20203.

Kahles F, Findeisen HM, Bruemmer D. 2014. Osteopontin: A novel regulator at the cross roads of inflammation, obesity and diabetes. Mol Metab. 3(4):384-393. Kerr G, Sheldon H, Chaikuad A, Alfano I, von Delft F, Bullock AN, Harris AL. 2015. A small molecule targeting alkl prevents notch cooperativity and inhibits functional angiogenesis. Angiogenesis. 18(2):209-217.

Klaning E, Christensen B, Sorensen ES, Vorup-Jensen T, Jensen JK. 2014. Osteopontin binds multiple calcium ions with high affinity and independently of phosphorylation status. Bone. 66:90- 95.

Klinthoopthamrong N, Chaikiawkeaw D, Phoolcharoen W, Rattanapisit K, Kaewpungsup P, Pavasant P, Hoven VP. 2020. Bacterial cellulose membrane conjugated with plant-derived osteopontin: Preparation and its potential for bone tissue regeneration. Int J Biol Macromol. 149:51-59.

Kon S, Ikesue M, Kimura C, Aoki M, Nakayama Y, Saito Y, Kurotaki D, Diao H, Matsui Y, Segawa T et al. 2008. Syndecan-4 protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin. J Exp Med. 205(1 ):25-33.

Liaw L, Birk DE, Balias CB, Whitsitt JS, Davidson JM, Hogan BL. 1998. Altered wound healing in mice lacking a functional osteopontin gene (sppl). J Clin Invest. 101(7): 1468-1478. Luukkonen J, Hilli M, Nakamura M, Ritamo I, Valmu L, Kauppinen K, Tuukkanen J, Lehenkari P. 2019. Osteoclasts secrete osteopontin into resorption lacunae during bone resorption. Histochem Cell Biol. l51(6):475-487.

McKee MD, Nanci A. 1996a. Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: Ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microsc Res Tech. 33(2): 141-164.

McKee MD, Nanci A. 1996b. Osteopontin deposition in remodeling bone: An osteoblast mediated event. J Bone Miner Res. 11(6):873-875.

Neron B, Menager H, Maufrais C, Joly N, Maupetit J, Letort S, Carrere S, Tuffery P, Letondal C. 2009. Mobyle: A new full web bioinformatics framework. Bioinformatics. 25(22):3005-3011. O'Regan A, Berman JS. 2000. Osteopontin: A key cytokine in cell-mediated and granulomatous inflammation. Int J Exp Pathol. 81(6):373-390.

Rattanapisit K, Abdulheem S, Chaikeawkaew D, Kubera A, Mason HS, Ma JK, Pavasant P, Phoolcharoen W. 2017. Recombinant human osteopontin expressed in nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells. Sci Rep. 7(1): 17358. Rittling SR, Matsumoto HN, McKee MD, Nanci A, An XR, Novick KE, Kowalski AJ, Noda M, Denhardt DT. 1998. Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro. J Bone Miner Res. 13(7): 1101-111 1.

Ross FP, Chappel J, Alvarez JI, Sander D, Butler WT, Farach-Carson MC, Mintz KA, Robey PG, Teitelbaum SL, Cheresh DA. 1993. Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption. J Biol Chem. 268( 13):9901-9907.

Singh A, Gill G, Kaur H, Amhmed M, Jakhu H. 2018. Role of osteopontin in bone remodeling and orthodontic tooth movement: A review. Prog Orthod. 19(1): 18.

Sodek J, Ganss B, McKee MD. 2000. Osteopontin. Crit Rev Oral Biol Med. 11(3):279-303. Tanabe N, Wheal BD, Kwon J, Chen HH, Shugg RP, Sims SM, Goldberg HA, Dixon SJ. 2011. Osteopontin signals through calcium and nuclear factor of activated t cells (nfat) in osteoclasts: A novel rgd-dependent pathway promoting cell survival. J Biol Chem. 286(46):39871-39881. Taylor KR, Gallo RL. 2006. Glycosaminoglycans and their proteoglycans: Host-associated molecular patterns for initiation and modulation of inflammation. FASEB J. 20(l):9-22.

Thumer PJ, Chen CG, lonova-Martin S, Sun L, Harman A, Porter A, Ager JW, 3rd, Ritchie RO, Alliston T. 2010. Osteopontin deficiency increases bone fragility but preserves bone mass. Bone. 46(6): 1564- 1573.

Townson SA, Martinez-Hackert E, Greppi C, Lowden P, Sako D, Liu J, Ucran JA, Liharska K, Underwood KW, Seehra J et al. 2012. Specificity and structure of a high affinity activin receptorlike kinase 1 (alkl) signaling complex. J Biol Chem. 287(33):27313-27325. van der Linden E, Venema P. 2007. Self-assembly and aggregation of proteins. Curr Opin Colloid In. 12(4-5): 158-165.

Wal PY, Kuo PC. 2008. Osteopontin: Regulation in tumor metastasis. Cancer Metastasis Rev. 27(l):103-118.

Wesson JA, Johnson RJ, Mazzali M, Beshensky AM, Stietz S, Giachelli C, Liaw L, Alpers CE, Couser WG, Kleinman JG et al. 2003. Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. J Am Soc Nephrol. 14(1): 139- 147.

Yang JM, Chen CC. 2004. Gemdock: A generic evolutionary method for molecular docking. Proteins. 55(2):288-304.

Zurick KM, Qin C, Bernards MT. 2013. Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate. J Biomed Mater Res A. 101 (6): 1571-1581.

Claims

1. A method of fabricating an implantable construct for a subject comprising providing a scaffold having a porous matrix, the scaffold being biocompatible to the subject; coating the scaffold with a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2; culturing cells capable of osteogenic differentiation on the coated scaffold; and acquiring the implantable construct upon attaining the osteogenic differentiation by the cultured cells.

2. The method of claim 1, wherein the peptide of amino acid sequence of SEQ ID No. 1 coated on the scaffold has a concentration ranged between 5 -20 ng/cm².

3. The method of claim 1, wherein the peptide of amino acid sequence of SEQ ID No. 2 coated on the scaffold has a concentration not exceeding 15 ng/cm²

4. The method of claim 1, wherein the cells is selected from any one or combination of periodontal ligament stem cells and bone marrow stromal cells.

5. The method of claim 1, wherein the osteogenic differentiation is by way of an interaction of a calcium binding domain in the peptide and ALK.-1 receptor of the cultured cells.

6. The method of claim I, wherein the subject is free of inhibitor of ALK-1 receptor.

7. The method of claim 1, wherein the culturing step is performed in the presence of a medium.

8. The method of claim 1, wherein the peptide is recombinant peptide produced from N. benthamiana.

9. A bio-engineered construct implantable to a subject comprising a scaffold having a porous matrix, the scaffold being biocompatible to the subject; a coating being deposited to the scaffold, the coating comprising a plurality of peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2; and cells capable of osteogenic differentiation being seeded and cultured on the scaffold substantially contacting with the coating, wherein the cells become osteogenic differentiated by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cultured cells.

10. The construct of claim 9, wherein the peptide of amino acid sequence of SEQ ID No. 1 coated on the scaffold has a concentration ranged between 5 -20 ng/cnr.

1 1. The construct of claim 9, wherein the cells is selected from any one or combination of periodontal ligament stem cells and bone marrow stromal cells.

12. The construct of claim 1 1 , wherein the cells are of autologous origin.

13. Use of a peptide having amino acid sequence as setting forth in SEQ ID No. 1 or SEQ ID No. 2 for initiating osteogenic differentiation of a cell selected from a group consisting of periodontal ligament stem cells and bone marrow stromal cells by way of an interaction of a calcium binding domain in the peptide and ALK-1 receptor of the cell.