Mineralised collagen—an artificial,extracellular bone matrix—improves osteogenic differentiation of bone marrow stromal cells

In the field of bone tissue engineering there is a high demand on bone graft materials which promote bone formation. By combination of collagen type I with nanocrystalline hydroxyapatite (HA) we generated a resorbable material which structure and composition is close to those of the extracellular bone matrix. This nanocomposit material was produced in a biomimetic process in which collagen fibril assembly and mineralisation with hydroxyapatite occur simultaneously. In this study the proliferation and osteogenic differentiation of human bone marrow-derived stromal cells (hBMSC) on membranes of biomimetically mineralised collagen type I was investigated. To this end, we optimised biochemical assays for determination of cell number and alkaline phosphatase activity corresponding to the special properties of this biomaterial. For cell experiments hBMSC were seeded on the mineralised collagen membranes and cultivated over 35 days, both in static and perfusion culture, in the presence and absence of dexamethasone, β-glycerophosphate and ascorbate. Compared to cells grown on tissue culture polystyrene we found attenuated proliferation rates, but markedly increased activity of alkaline phosphatase on the mineralised collagen indicating its promoting effect on the osteogenic differentiation of hBMSC. Therefore this bone-like material may act as a suitable artificial extracellular matrix for bone tissue engineering. Perfusion of the 2D cell matrix constructs with cell culture medium did not improve proliferation and osteogenic differentiation of the hBMSC. Anne Bernhardt and Anja Lode contributed equally to this paper     

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on growth and osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cell differentiation of osteoblasts is triggered by a series of signaling processes including integrin and bone morphogenetic protein (BMP), which therefore act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in an artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffold. Matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were seeded with human mesenchymal stem cells (hMSC) and cultivated over a period of 22 days, either under growth or osteoinductive conditions. During the course of culture, gene expression of alkaline phosphatase (ALP), osteocalcin (OC) and collagen I (COL-I) as well as Smad5 and focal adhesion kinase (FAK), two signal transduction molecules involved in BMP-2 or integrin signaling were analyzed. Furthermore, calcium and collagen I deposition, as well as cell densities and proliferation, were determined using fluorescence microscopy. The incorporation of BMP-2 into PLLA-collagen type I nanofibers resulted in a decrease in diameter as well as pore sizes of the scaffold. Mesenchymal stem cells showed better adherence and a reduced proliferation on BMP-containing scaffolds. This was accompanied by an increase in gene expression of ALP, OC and COL-I. Furthermore the presence of BMP-2 resulted in an upregulation of FAK, while collagen had an impact on the gene expression of Smad5. Therefore these different strategies can be combined in order to enhance the osteoblast differentiation of hMSC on PLLA based nanofiber scaffold. By doing this, different signal transduction pathways seem to be up regulated.     

Effect of direct RGD incorporation in PLLA nanofibers on growth and osteogenic differentiation of human mesenchymal stem cells

The aim of this study was to functionalize synthetic poly-(l-lactic) (PLLA) nanofibers by direct incorporation of cRGD, in order to promote adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. The cRGD was incorporated into PLLA nanofibers either by emulsion [PLLA-cRGD (d)] or suspension [PLLA-cRGD (s)]. Matrices were seeded with hMSC and cultivated over a period of 28 days under growth conditions and analyzed during the course. Although the mode of incorporation resulted in different distributions of the RGD peptide, it had no impact on the fiber characteristics when compared to corresponding unblended PLLA control fibers. However, hMSC showed better adherence on PLLA-cRGD (d). Nevertheless, this advantage was not reflected during the course of cultivation. Furthermore, the PLLA-cRGD (s) fibers mediated the osteogenic potential of collagen (determined as the expression and deposition of collagen and osteocalcin) to some extent. Further studies are needed in order to optimize the RGD distribution and concentration.     

Electrospun polyvinyl alcohol-collagen-hydroxyapatite nanofibers: a biomimetic extracellular matrix for osteoblastic cells

The failure of prosthesis after total joint replacement is due to the lack of early implant osseointegration. In this study polyvinyl alcohol-collagen-hydroxyapatite (PVA-Col-HA) electrospun nanofibrous meshes were fabricated as a biomimetic bone-like extracellular matrix for the modification of orthopedic prosthetic surfaces. In order to reinforce the PVA nanofibers, HA nanorods and Type I collagen were incorporated into the nanofibers. We investigated the morphology, biodegradability, mechanical properties and biocompatibility of the prepared nanofibers. Our results showed these inorganic-organic blended nanofibers to be degradable in vitro. The encapsulated nano-HA and collagen interacted with the PVA content, reinforcing the hydrolytic resistance and mechanical properties of nanofibers that provided longer lasting stability. The encapsulated nano-HA and collagen also enhanced the adhesion and proliferation of murine bone cells (MC3T3) in vitro. We propose the PVA-Col-HA nanofibers might be promising modifying materials on implant surfaces for orthopedic applications.     

Tailoring material properties of a nanofibrous extracellular matrix derived hydrogel

In the native tissue, the interaction between cells and the extracellular matrix (ECM) is essential for cell migration, proliferation, differentiation, mechanical stability, and signaling. It has been shown that decellularized ECMs can be processed into injectable formulations, thereby allowing for minimally invasive delivery. Upon injection and increase in temperature, these materials self-assemble into porous gels forming a complex network of fibers with nanoscale structure. In this study we aimed to examine and tailor the material properties of a self-assembling ECM hydrogel derived from porcine myocardial tissue, which was developed as a tissue specific injectable scaffold for cardiac tissue engineering. The impact of gelation parameters on ECM hydrogels has not previously been explored. We examined how modulating pH, temperature, ionic strength, and concentration affected the nanoscale architecture, mechanical properties, and gelation kinetics. These material characteristics were assessed using scanning electron microscopy, rheometry, and spectrophotometry, respectively. Since the main component of the myocardial matrix is collagen, many similarities between the ECM hydrogel and collagen gels were observed in terms of the nanofibrous structure and modulation of properties by altering ionic strength. However, variation from collagen gels was noted for the gelation temperature along with varied times and rates of gelation. These discrepancies when compared to collagen are likely due to the presence of other ECM components in the decellularized ECM based hydrogel. These results demonstrate how the material properties of ECM hydrogels could be tailored for future in vitro and in vivo applications.     

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.     

Biomimetic synthesis and biocompatibility evaluation of carbonated apatites template-mediated by heparin

Biomimetic synthesis of carbonated apatites with good biocompatibility is a promising strategy for the broadening application of apatites for bone tissue engineering. Most researchers were interested in collagen or gelatin-based templates for synthesis of apatite minerals. Inspired by recent findings about the important role of polysaccharides in bone biomineralization, here we reported that heparin, a mucopolysaccharide, was used to synthesize carbonated apatites in vitro. The results indicated that the Ca/P ratio, carbon content, crystallinity and morphology of the apatites varied depending on the heparin concentration and the initial pH value. The morphology of apatite changed from flake-shaped to needle-shaped, and the degree of crystallinity decreased with the increasing of heparin concentration. Biocompatibility of the apatites was tested by proliferation and alkaline phosphatase activity of MC3T3-E1 cells. The results suggested that carbonated apatites synthesized in the presence of heparin were more favorable to the proliferation and differentiation of MC3T3-E1 cells compared with traditional method. In summary, the heparin concentration and the initial pH value play a key role in the chemical constitution and morphology, as well as biological properties of apatites. These biocompatible nano-apatite crystals hold great potential to be applied as bioactive materials for bone tissue engineering.     

神经脱细胞基质凝胶/BMP-2/脱钙骨复合材料的制备及性能

以神经脱细胞基质(NAM)凝胶作为载体,在多孔脱钙骨内表面负载BMP-2,制备新型复合骨修复材料。采用扫描电镜观察材料微观结构,结果表明脱钙骨基质(DBM)复合负载BMP-2的脱细胞基质凝胶后有利于细胞粘附;力学检测结果显示脱钙骨未复合神经脱细胞基质凝胶时,材料的抗压强度为(1.04±0.44)MPa,脱钙骨复合神经脱细胞基质凝胶之后,抗压强度为(1.00±0.30)MPa,材料复合凝胶前后力学性能改变不明显;模拟体内环境,检测不同时间点复合材料中的BMP-2缓释性能,复合材料可缓慢释放BMP-2至少一个月;在材料内部种植MC3TE-E1细胞,共培养14天,CCK-8、碱性磷酸酶检测表明负载BMP-2的神经脱细胞基质凝胶复合脱钙骨材料能够促进细胞增殖和成骨分化。神经脱细胞基质凝胶/BMP-2/钙骨复合材料具有潜在的骨修复应用前景。     

In vitro Response of Human Mesenchymal Stromal Cells to Titanium Coated Peek Films and Their Suitability for Magnetic Resonance Imaging

Medical imaging is an important tool for the post-operative checkup of an accurate position of an implant as well as for monitoring the integration in the adjacent tissue that may influence the success of a medical device.Unfortunately,the possibility to use imaging methods is associated with the implant material and all the established metallic materials for surgery do not show a proper "imaging compatibility".The present study is a combined investigation of the in vitro response to human mesenchymal stromal cells(hMSC) and magnetic resonance imaging(MRI) compatibility of the potential material combination polyetheretherketone/titanium(PEEK/Ti) for medical devices.Because of the advantageous imaging properties and the mechanical and chemical stability,PEEK becomes more and more an alternative to common metallic implant materials like titanium or cobalt-chrome.However,PEEK is a bioinert material having a limited ability for direct bone incorporation.Due to its excellent biocompatibility,Ti was chosen as coating material to enhance the cellular response.The result is a combination with advantageous properties:the magnetic susceptibility and elastic modulus close to bone,corrosion resistance and mechanical flexibility of PEEK and the excellent biocompatibility of titanium.The appearance of metal-related artifact was discussed in electrical resistivity and magnetic susceptibility.Therefore,two titanium coatings have been investigated:a complete coating and a structured surface avoiding surface conductivity.To determine the in vitro biocompatibility,the cell responses were assessed in terms of the overall morphology of the hMSC and their cell area distribution,proliferation,osteogenic differentiation and mineral deposition.The cellular stress was evaluated by the prostaglandin E_2 level.The bonded materials both produced no disturbing artifacts in magnetic resonance imaging.Compared to the pure PEEK material,the titanium coated specimens showed an enhanced biocompatibility,which is indicated by a higher cell number,larger activity of the enzyme tissue non-specific alkaline phosphatase and therefore a greater amount of deposited calcium and phosphate.The results on bare PEEK are accompanied with a higher cellular stress level,which is indicated by prostaglandin E_2.     

Implants of polyanionic collagen matrix in bone defects of ovariectomized rats

In recent years, there has been a great interest in the development of biomaterials that could be used in the repair of bone defects. Collagen matrix (CM) has the advantage that it can be modified chemically to improve its mechanical properties. The aim of the present study was to evaluate the effect of three-dimensional membranes of native or anionic (submitted to alkaline treatment for 48 or 96 h) collagen matrix on the consolidation of osteoporosis bone fractures resulting from the gonadal hormone alterations caused by ovariectomy in rats subjected to hormone replacement therapy. The animals received the implants 4 months after ovariectomy and were sacrificed 8 weeks after implantation of the membranes into 4-mm wide bone defects created in the distal third of the femur with a surgical bur. Macroscopic analysis revealed the absence of pathological alterations in the implanted areas, suggesting that the material was biocompatible. Microscopic analysis showed a lower amount of bone ingrowth in the areas receiving the native membrane compared to the bone defects filled with the anionic membranes. In ovariectomized animals receiving anionic membranes, a delay in bone regeneration was observed mainly in animals not subjected to hormone replacement therapy. We conclude that anionic membranes treated with alkaline solution for 48 and 96 h presented better results in terms of bone ingrowth.     

Response of primary fibroblasts and osteoblasts to plasma treated polyetheretherketone (PEEK) surfaces

Polyetheretherketone (PEEK) is a synthetic polymer with suitable biomechanical and stable chemical properties, which make it attractive for use as an endoprothetic material and for ligamentous replacement. However, chemical surface inertness does not account for a good interfacial biocompatibility, and PEEK requires a surface modification prior to its application in vivo.In the course of this experimental study we analyzed the influence of plasma treatment of PEEK surfaces on the cell proliferation and differentiation of primary fibroblasts and osteoblasts. Further we examined the possibility of inducing microstructured cell growth on a surface with plasma-induced chemical micropatterning.We were able to demonstrate that the surface treatment of PEEK with a low-temperature plasma has significant effects on the proliferation of fibroblasts. Depending on the surface treatment, the proliferation rate can either be stimulated or suppressed. The behavior of the osteoblasts was examined by evaluating differentiation parameters.By detection of alkaline phosphatase, collagen I, and mineralized extracellular matrix as parameters for osteoblastic differentiation, the examined materials showed results comparable to commercially available polymer cell culture materials such as tissue culture polystyrene (TCPS). Further microstructured cell growth was produced successfully on micropatterned PEEK foils, which could be a future tool for bioartificial systems applying the methods of tissue engineering.These results show that chemically inert materials such as PEEK may be modified specifically through the methods of plasma technology in order to improve biocompatibility.The first two authors share first authorship.     

Preparation and characterization of mesoporous bioactive glass/polycaprolactone nanofibrous matrix for bone tissues engineering

A polycaprolactone (PCL) nanofibrous composite matrix having mesoporous bioactive glass nanoparticles (MBG) was fabricated using the electrospinning method, and the microstructural, physical and biological properties of the composite matrix were characterized. The fiber diameters of PCL, 5?% MBG/PCL (5?M-PCL) and 10?% MBG/PCL (10?M-PCL) were 575?±?162?nm, 312?±?134?nm and 321?±?144?nm, respectively. The bioactivity of the composite matrix was evaluated by soaking the matrix in 1.5× simulated body fluid; the MBG/PCL matrix showed a better biomineralization capability than did the PCL matrix. The biological performance of the PCL and the MBG/PCL were evaluated using an in vitro culture of MG63 osteoblast-like cells. We found that the cell attachment and proliferation rates were significantly higher on the 10?M-PCL than on the PCL. Moreover, the expression of several genes, including ANX-V, type I collagen and OCN, ALP activity, the deposition of calcium, and the BSP protein, were also significantly higher on 10?M-PCL than PCL. These results indicated that MBG/PCL has the ability to support cell attachment, growth, and differentiation and can also yield high bioactivity. Therefore, MBG/PCL could be potentially applied in bone implants.     

Engineered articular cartilage: influence of the scaffold on cell phenotype and proliferation

Articular cartilage defects do not heal. Biodegradable scaffolds have been studied for cartilage engineering in order to implant autologous chondrocytes and help cartilage repair. We tested some new collagen matrices differing in collagen type, origin, structure and methods of extraction and purification, and compared the behavior of human chondrocytes cultured on them. Human chondrocytes were grown for three weeks on four different equine type I collagen matrices, one type I, III porcine collagen matrix and one porcine type II collagen matrix. After 21 days, samples were subjected to histochemical, immunohistochemical and histomorphometric analysis to study phenotype expression and cell adhesion. At 7, 14 and 21 days cell proliferation was studied by incorporation of [3H]-thymidine. Our data evidence that the collagen type influences cell morphology, adhesion and growth; indeed, cellularity and rate of proliferation were significantly higher and cells were rounder on the collagen II matrix than on either of the collagen I matrices. Among the collagen I matrices, we observed a great variability in terms of cell adhesion and proliferation. The present study allowed us to identify one type I collagen matrix and one type II collagen matrix that could be usefully employed as a scaffold for chondrocyte transplantation.     

Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties

Tissue engineering scaffolds encourage cell proliferation whilst degrading to facilitate tissue regeneration. Their mechanical properties therefore change, decreasing due to scaffold degradation and increasing due to extracellular matrix deposition. This work compares the changing properties of collagen scaffolds incubated in culture medium, with and without human tenocytes, in order to investigate the relationship between degradation and tenocyte proliferation. The material properties of scaffolds are compared over 26 days using mechanical testing, differential scanning calorimetry, infra-red spectroscopy, and histology and biochemical assays. For medium-only scaffolds, the mechanical properties decrease rapidly, while culture medium sulfhydryl content increases significantly, with no significant changes in the denaturation temperature of scaffold collagen content. Conversely, the mechanical properties and collagen content of tenocyte-seeded scaffolds increase significantly while culture medium sulfhydryl content decreases and denaturation temperature remains the same. These results indicate that tenocytes proliferation both reduces the degradation of collagen scaffolds incubated in culture medium and produces scaffolds with improved properties.     

Loading of VEGF to the heparin cross-linked demineralized bone matrix improves vascularization of the scaffold

Deficient vascularization is one of the prominent shortcomings of porous tissue-engineering scaffolds, which results in insufficient oxygen and nutrients transportation. Here, heparin cross-linked demineralized bone matrices (HC-DBM) pre-loaded with vascular endothelial growth factor (VEGF) were designed to promote cells and new microvessels invasion into the matrices. After being chemical crosslinked with heparin by N-hydroxysuccinimide and N-(3-di-methylaminopropyl)-N’-ethylcarbodiimide, the scaffold could bind more VEGF than the non-crosslinked one and achieve localized and sustained delivery. The biological activity of VEGF binding on heparinized collagen was demonstrated by promoting endothelial cells proliferation. Evaluation of the angiogenic potential of heparinized DBM loaded with VEGF was further investigated by subcutaneous implantation. Improved angiogenesis of heparinized DBM loaded with VEGF was observed from haematoxylin-eosin staining and immunohistochemistry examination. The results demonstrated that heparin cross-linked DBM binding VEGF could be a useful strategy to stimulate cells and blood vessels invasion into the scaffolds.     

Parallel high-resolution confocal Raman SEM analysis of inorganic and organic bone matrix constituents.

In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS-SEM revealed that early in vitro-formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS-SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.     

Nanophase hydroxyapatite and poly(lactide-co-glycolide) composites promote human mesenchymal stem cell adhesion and osteogenic differentiation in vitro

Human mesenchymal stem cells (hMSCs) typically range in size from 10 to 50 μm and proteins that mediate hMSC adhesion and differentiation usually have a size of a few nanometers. Nanomaterials with a feature size smaller than 100 nm have demonstrated the unique capability of promoting osteoblast (bone forming cell) adhesion and long-term functions, leading to more effective bone tissue regeneration. For new bone deposition, MSCs have to be recruited to the injury or disease sites and then differentiate into osteoblasts. Therefore, designing novel nanomaterials that are capable of attracting MSCs and directing their differentiation is of great interest to many clinical applications. This in vitro study investigated the effects of nanophase hydroxyapatite (nano-HA), nano-HA/poly(lactide-co-glycolide) (PLGA) composites and a bone morphogenetic protein (BMP-7) derived short peptide on osteogenic differentiation of hMSCs. The short peptide was loaded by physical adsorption to nano-HA or by dispersion in nanocomposites and in PLGA to determine their effects on hMSC adhesion and differentiation. The results showed that the nano-HA/PLGA composites promoted hMSC adhesion as compared to the PLGA controls. Moreover, nano-HA/PLGA composites promoted osteogenic differentiation of hMSCs to a similar extent with or without the presence of osteogenic factors in the media. In the MSC growth media without the osteogenic factors, the nanocomposites supported greater calcium-containing bone mineral deposition by hMSC than the BMP-derived short peptide alone. The nanocomposites provided promising alternatives in controlling the adhesion and differentiation of hMSCs without osteogenic factors from the culture media, and, thus, should be further studied for clinical translation and the development of novel nanocomposite-guided stem cell therapies.     

Determination of bone mineral volume fraction using impedance analysis and Bruggeman model

Measurements by impedance spectroscopy and Bruggeman effective medium approximation model were employed in order to determine the mineral volume fraction of dry bone. This approach assumes that two or more phases are present into the composite: the matrix (environment) and the other ones are inclusion phases. A fragment of femur diaphysis dense bone from a young pig was investigated in its dehydrated state. Measuring the dielectric properties of bone and its main components (hydroxyapatite and collagen) and using the Bruggeman approach, the mineral volume filling factor was determined. The computed volume fraction of the mineral volume fraction was confirmed by a histogram test analysis based on the SEM microstructures. In spite of its simplicity, the method provides a good approximation for the bone mineral volume fraction. The method which uses impedance spectroscopy and EMA modeling can be further developed by considering the conductive components of the bone tissue as a non-invasive in situ impedance technique for bone composition evaluation and monitoring.     

Injectable collagen/α-tricalcium phosphate cement: collagen–mineral phase interactions and cell response

A bone inspired material was obtained by incorporating collagen in the liquid phase of an α-tricalcium phosphate cement, either in solubilized or in fibrilized form. This material was able to set in situ, giving rise to a calcium deficient hydroxyapatite (CDHA)/collagen composite. The morphology and distribution of collagen in the composite was shown to be strongly affected by the collagen pre-treatment. The interactions between collagen and the inorganic phase were assessed by FTIR. A red shift of the amide I band was indicative of calcium chelation by the collagen carbonyl groups. The rate of CDHA formation was not affected when diluted collagen solutions (1 mg/ml) were used, whereas injectability improved. The presence of solubilized collagen, even in low amount (1 %), increased cell adhesion and proliferation on the composites. Still in the absence of osteogenic medium, significant ALP activity was detected both in the inorganic and the collagen-containing cements. The maximum ALP activity was advanced in the collagen-containing cement as compared to the inorganic cement.     

Post implantation fate of adipogenic induced mesenchymal stem cells on Type I collagen scaffold in a rat model

Regenerative medicine via its application in soft tissue reconstruction through novel methods in adipose tissue engineering (ATE) has gained remarkable attention and investment despite simultaneous reports on clinical incidence of graft resorption and impaired vascularization. The underlying malaise here once identified may play a critical role in optimizing implant function. Our work attempts to determine the fate of donor cells and the implant in recipient micro environment using adipose-derived mesenchymal stem cells (ASCs) on a type I collagen sponge, an established scaffold for ATE. Cell components within the construct were identified 21 days post implantation to delineate cell survival, proliferation & terminal roles in vivo. ASC’s are multipotent, while collagen type I is a natural extra cellular matrix component. Commercially available bovine type I collagen was characterized for its physiochemical properties and cyto-compatibility. Nile red staining of induced ASCs identified red globular structures in cell cytoplasm indicating oil droplet accumulation. Similarly, in vivo implantation of the cell seeded collagen construct in rat model for 21 days in the dorsal muscle, showed genesis of chicken wire network of fat-like cells, which was demonstrated histologically using a variety of staining techniques. Furthermore, fluorescent in situ hybridization (FISH) technique established the efficiency of transplantation wherein the male donor cells with labeled Y chromosome was identified 21 days post implantation from female rat model. Retrieved samples at 21 days indicated adipogenesis in situ, with donor cells highlighted via FISH. The study provides an insight to stem cells in ATE from genesis to functionalization.