Pre-culture period of mesenchymal stem cells in osteogenic media influences their in vivo bone forming potential

The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different time periods (4, 10, and 16 days) in tissue culture plates (TCP), seeded on sintered titanium fiber meshes without the extracellular matrix (ECM) generated in vitro, and implanted in the rat cranium after 12 h. Thirty two adult Fisher rats received the implants, divided in four groups. Three groups were implanted with cells cultured for 4, 10, or 16 days in osteogenic media and at that time their alkaline phosphatase activity and mineral deposition denoted that they were at different stages of their osteoblastic maturation (undifferentiated MSC, committed, and mature Osteoblasts, respectively). MSCs cultured without osteogenic media for 6 days were used as controls. The constructs were retrieved 4 weeks later and processed for histomorphometric analysis. Implants seeded with cells that have been cultured with osteogenic media for only 4 days revealed the highest bone formation. The lowest bone formation was obtained with the implants seeded with MSCs cultured for 16 days in the presence of osteogenic media. The results of this study suggested that the in vitro pre-culture period of MSCs is a critical factor for their ability to regenerate bone when implanted to an orthotopic site.     

Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds

In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 m fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 × 10⁵ cells in 300 l, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.     

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.     

Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.     

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh

The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh.     

Three-dimensional culture of differentiating marrow stromal osteoblasts in biomimetic poly(propylene fumarate-co-ethylene glycol)-based macroporous hydrogels

This study assesses the ability of biomimetic poly(propylene fumarate-co-ethylene glycol)-based hydrogels to sustain the differentiation of marrow stromal cells (MSCs) to the osteoblastic phenotype and to produce a mineralized matrix in vitro. Macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) with and without covalently linked RGD cell-adhesive peptide were synthesized and seeded with rat MSCs suspended in media or in a type I collagen solution. Cells suspended in media were found to adhere to RGD-modified but not to unmodified hydrogels. Cells suspended in a collagen solution were entrapped after collagen gelation and proliferated independent of the peptide modification of the hydrogel. Hydrogel modification with RGD peptide was sufficient to allow for the adhesion and differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture supplements. MSCs seeded with a collagen gel onto RGD-modified macroporous hydrogels after 28 days of culture showed a significant increase in cell numbers, from 15,200 +/- 2,000 to 208,600 +/- 69,700 cells (p < 0.05). Moreover, significant calcium deposition was apparent after 28 days of culture in RGD-modified hydrogels for cells suspended in a collagen gel in comparison to cells suspended in media, 3.47 +/- 0.26 compared to 0.82 +/- 0.20 mg Ca(2+) per scaffold (p < 0.05). Confocal microscopy revealed that MSCs suspended in a collagen gel and cultured on RGD-modified hydrogels for 28 days were adhered to the surface of the hydrogel while MSCs suspended in a collagen gel and cultured on unmodified hydrogels were located within the pores of and not in direct contact with the hydrogel surface. The results demonstrate that these biomimetic hydrogels facilitate the adhesion and support the differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture media.     

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The interplay of bone-like extracellular matrix and TNF-α signaling on in vitro osteogenic differentiation of mesenchymal stem cells

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing na?ve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration.     

The effect of differentiation stage of amniotic fluid stem cells on bone regeneration

Bone tissue engineering strategies require cells with high proliferative and osteogenic potential as well as a suitable scaffold to support the development of these as they form new bone tissue. In this study, we evaluated whether the differentiation stage of amniotic fluid stem cells (AFSC) could enhance the regeneration of critical sized femoral defects in a rat model. For this purpose, AFSC were seeded onto a starch-poly(ε-caprolactone) (SPCL) scaffold and were cultured in vitro in osteogenic culture media for different periods of time in order to obtain: i) undifferentiated cells, ii) cells committed to the osteogenic phenotype and iii) "osteoblast-like" cells. In vitro results indicate that AFSC were considered to be osteogenically committed by the end of week 2 and osteoblastic-like after week 3 in culture. Constructs composed of AFSC-SPCL scaffolds from each differentiation stage were implanted into critical sized femoral defects. The quality of new tissue formed in the defects was evaluated based on micro-CT imaging and histological analysis of constructs retrieved at 4 and 16 weeks after implantation. In vivo formation of new bone was observed under all conditions. However, the most complete repair of the defect was observed after 16 weeks in the animals receiving the SPCL scaffolds seeded with osteogenically committed AFSC. Furthermore, the presence of blood vessels was noted in the inner sections of the scaffolds suggests that these cells could potentially be used to induce bone regeneration and angiogenesis in non-union bone defects.     

Proliferation and differentiation of human mesenchymal stem cell encapsulated in polyelectrolyte complexation fibrous scaffold

A biofunctional scaffold was constructed with human mesenchymal stem cells (hMSCs) encapsulated in polyelectrolyte complexation (PEC) fibers. Human MSCs were either encapsulated in PEC fibers and constructed into a fibrous scaffold or seeded on PEC fibrous scaffolds. The proliferation, chondrogenic and osteogenic differentiation of the encapsulated and seeded hMSCs were compared for a culture period of 5.5 weeks. Gene expression and extracellular matrix production showed evidences of chondrogenesis and osteogenesis in the cell-encapsulated scaffolds and cell-seeded scaffolds when the samples were cultured in the chondrogenic and osteogenic differentiation media, respectively. However, better cell proliferation and differentiation were observed on the hMSC-encapsulated scaffolds compared to the hMSC-seeded scaffolds. The study demonstrated that the cell-encapsulated PEC fibers could support proliferation and chondrogenic and osteogenic differentiation of the encapsulated-hMSCs. Together with our previous works, which demonstrated the feasibility of PEC fiber in controlled release of drug, protein and gene delivery, the reported PEC fibrous scaffold system will have the potential in composing a multi-component system for various tissue-engineering applications.     

Modulation of osteogenic properties of biodegradable polymer/extracellular matrix scaffolds generated with a flow perfusion bioreactor

In this study, composite scaffolds consisting of both synthetic and natural components with controllable properties were generated by incorporating mineralized extracellular matrix (ECM) and electrospun poly(ε-caprolactone) (PCL) microfiber scaffolds. Mesenchymal stem cells (MSCs) were cultured on PCL scaffolds under flow perfusion conditions with culture medium supplemented with dexamethasone to investigate the effect of culture duration on mineralized extracellular matrix deposition. MSCs differentiated down the osteogenic lineage and produced extracellular matrix with different compositions of mineral, collagen, and glycosaminoglycan with distinct morphologies at various stages of osteogenesis. To determine whether the presence and maturity of mineralized extracellular matrix influences osteogenic differentiation in vitro, PCL/ECM constructs were decellularized to yield PCL/ECM composite scaffolds that were subsequently seeded with MSCs and cultured in the absence of dexamethasone. The presence of mineralized matrix reduced cellular proliferation while stimulating alkaline phosphatase activity with increasing amounts of calcium deposition over time. PCL/ECM composite scaffolds containing the most mature mineralized matrix resulted in the most rapid increase and highest levels of alkaline phosphatase activity and calcium deposition compared to all other scaffold groups. Therefore, we demonstrate that mineralized extracellular matrix generated under controlled flow perfusion conditions can impart osteogenic properties to an osteoconductive polymer scaffold, and that the maturity of this matrix influences osteogenic differentiation in vitro, even in the absence of dexamethasone.     

Effect of hydrogel porosity on marrow stromal cell phenotypic expression

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.     

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.     

In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold

The objective of this study was to assess bone formation from mesenchymal stem cells (MSCs) on a novel nanofibrous scaffold in a rat model. A highly porous, degradable poly(epsilon-caprolactone) (PCL) scaffold with an extracellular matrix-like topography was produced by electrostatic fiber spinning. MSCs derived from the bone marrow of neonatal rats were cultured, expanded, and seeded on the scaffolds. The cell-polymer constructs were cultured with osteogenic supplements in a rotating bioreactor for 4 weeks, and subsequently implanted in the omenta of rats for 4 weeks. The constructs were explanted and characterized by histology, immunohistochemistry, and scanning electron microscopy. The constructs maintained the size and shape of the original scaffolds. Morphologically, the constructs were rigid and had a bone-like appearance. Cells and extracellular matrix (ECM) formation were observed throughout the constructs. In addition, mineralization and type I collagen were also detected. This study establishes the ability to develop bone grafts on electrospun nanofibrous scaffolds in a well-vascularized site using MSCs.     

Osteogenic differentiation of bone marrow stromal cells on poly(ε-caprolactone) nanofiber scaffolds

Nanofiber poly(ε-caprolactone) (PCL) scaffolds were fabricated by electrospinning, and their ability to enhance the osteoblastic behavior of marrow stromal cells (MSCs) in osteogenic media was investigated. MSCs were isolated from Wistar rats and cultured on nanofiber scaffolds to assess short-term cytocompatibility and long-term phenotypic behavior. Smooth PCL substrates were used as control surfaces. The short-term cytocompatibility results indicated that nanofiber scaffolds supported greater cell adhesion and viability compared with control surfaces. In osteogenic conditions, MSCs cultured on nanofiber scaffolds also displayed increased levels of alkaline phosphatase activity for 3 weeks of culture. Calcium phosphate mineralization was substantially accelerated on nanofiber scaffolds compared to control surfaces as indicated through von Kossa and calcium staining, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Increased levels of intra- and extracellular levels of osteocalcin and osteopontin were observed on nanofiber scaffolds using immunofluorescence techniques after 3 weeks of culture. These results demonstrate the enhanced tissue regeneration property of nanofiber scaffolds, which may be of potential use for engineering osteogenic scaffolds for orthopedic applications.     

Bilayered constructs aimed at osteochondral strategies: the influence of medium supplements in the osteogenic and chondrogenic differentiation of amniotic fluid-derived stem cells

The development of osteochondral tissue engineered interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints. This study reports the development of a bilayered scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone (SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were cultured for 1 or 2 weeks in an osteochondral-defined culture medium containing both osteogenic and chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression were assessed within the constructs in order to determine the influence of the osteochondral differentiation medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced a protein-rich, mineralized extracellular matrix (ECM) for up to 2 weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces. In addition, constructs generated from agarose-SPCL bilayered scaffolds containing pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased joints.     

Evaluation of extracellular matrix formation in polycaprolactone and starch-compounded polycaprolactone nanofiber meshes when seeded with bovine articular chondrocytes

Cartilage defects are a major health problem. Tissue engineering has developed different strategies and several biomaterial morphologies, including natural-based ones, for repairing these defects. We used electrospun polycaprolactone (PCL) and starch-compounded PCL (SPCL) nanofiber meshes to evaluate extracellular matrix (ECM) formation by bovine articular chondrocytes (BACs). The main aim of this work was to evaluate the suitability of PCL and SPCL nanofiber meshes in chondrocyte cultures, and their capability to produce ECM when seeded onto these nanostructured materials. The effect of culture conditions (static vs dynamic) on ECM formation was also assessed. BACs were seeded onto PCL and SPCL nanofiber meshes using a dynamic cell-seeding procedure and cultured under static or dynamic conditions for 4 weeks. Constructs were characterized using scanning electron microscopy, histology, immunolocalization of collagen types I and II, and glycosaminoglycan (GAG) quantification. Results show an extensive cell colonization of the entire nanofiber mesh, for both materials, and that chondrocytes presented typical spherical morphology. Some degree of cell infiltration inside the nanofiber meshes was noticeable for both materials. ECM formation and GAG were detected throughout the materials, evidencing typical construct maturation. PCL and SPCL nanofiber meshes are suitable as supports for ECM formation and therefore are adequate for cartilage tissue-engineering approaches.     

Effect of temporally patterned TNF-α delivery on in vitro osteogenic differentiation of mesenchymal stem cells cultured on biodegradable polymer scaffolds

Recent insight into the critical role of pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), in bone regeneration has heralded a new direction in the design of tissue engineering constructs. Previous studies have demonstrated that continuous delivery of 50?ng/ml TNF-α to mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) biodegradable electrospun poly(?-caprolactone) (PCL) microfiber meshes stimulates mineralized matrix deposition, a marker of osteogenic differentiation. Since TNF-α exhibits a biphasic pattern of expression following bone fracture in vivo, this study aimed to investigate the effects of temporal patterns of TNF-α delivery on in vitro osteogenic differentiation of MSCs cultured on 3D electrospun PCL scaffolds. MSCs were cultured for 16?days and exposed to continuous, early, intermediate, or late TNF-α delivery. To further elucidate the effects of TNF-α on osteogenic differentiation, the study design included MSCs precultured both in the presence and absence of typically required osteogenic supplement dexamethasone. Mineralized matrix deposition was not observed in constructs with dexamethasone-naïve MSCs, suggesting that TNF-α is not sufficient to trigger in vitro osteogenic differentiation of MSCs. For MSCs precultured with dexamethasone, TNF-α suppressed alkaline phosphatase activity, an early marker of osteogenic differentiation, and stimulated mineralized matrix deposition, a late stage marker of MSC osteogenic differentiation. By elucidating the impact of temporal variations in TNF-α delivery on MSC osteogenic differentiation, our results offer insight into the regenerative mechanism of TNF-α and provide the design parameters for a novel tissue engineering strategy that rationally controls TNF-α signaling to stimulate bone regeneration.