Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation

The osteogenic potential of mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA) or poly(caprolactone) (PCL), two widely used polymeric biomaterials that have been reported to differentially support osteogenic differentiation, was compared in these studies. Here we report that MSCs cultured in 3-D PLGA scaffolds for up to 5 weeks significantly upregulate osteocalcin gene expression levels. By contrast, osteocalcin expression was markedly downregulated in 3-D PCL-based constructs over the same time course. We hypothesized that differential adsorption of extracellular matrix (ECM) proteins present in serum-containing culture medium and subsequent differences in integrin-mediated adhesion are responsible for these differences, and tested this hypothesis using thin (2-D) polymeric films. Supporting this hypothesis, significant amounts of fibronectin and vitronectin deposited onto both materials in serum-containing osteogenic media, with type-I collagen present in lower amounts. Adhesion-blocking studies revealed that MSCs adhere to PCL primarily via vitronectin, while type-I collagen mediates their attachment to PLGA. These adhesive mechanisms correlated with higher levels of alkaline phosphatase (ALP) activity after 2 weeks of monolayer culture on PLGA versus PCL. These data suggest that the initial adhesion of MSCs to PLGA via type-I collagen fosters osteogenesis while adhesion to PCL via vitronectin does not, and stress the need for an improved molecular understanding of cell-ECM interactions in stem cell-based therapies.     

Conditioned Media Enhance Osteogenic Differentiation on Poly(L-lactide-co-ε-caprolactone)/Hydroxyapatite Scaffolds and Chondrogenic Differentiation in Alginate

The biochemical factors that regulate cell proliferation and differentiation can provide a means of optimizing culture conditions to develop a tissue-engineered osteochondral construct. Thus, the objectives of this study were to determine the effects of chondrocyte conditioned medium (CM) on the osteogenic differentiation of mesenchymal stem cells (MSCs) cultured on poly(L-lactide-co-ε-caprolactone)/hydroxyapatite (PLA/PCL/HAP) scaffolds and to determine the effect of osteoblast CM on the chondrogenic differentiation of MSCs cultured in alginate. In addition, the biomaterial's effect on MSC differentiation was also investigated. MSCs were grown in two groups: (1) on porous PLA/PCL/HAP scaffolds in osteogenic differentiation medium or (2) encapsulated in alginate in chondrogenic differentiation medium. CM was taken from one group and administered to the 'opposite' group in volumetric concentrations of 25% or 50% at each medium change. The osteogenic group samples that were administered chondrocyte CM showed higher alkaline phosphatase activity than the controls that were not administered CM. Additionally, the cells that were given chondrocyte CM had higher osteocalcin and sialoprotein expression than the controls. Samples in the chondrogenic group that were administered osteoblast CM at a volumetric concentration of 50% produced more sGAG than the controls. The aggrecan and Sox9 expression was significantly higher in the samples given 50% CM as compared to the controls. The study also showed that culturing cells in alginate, without differentiation medium, can produce similar levels of differentiation as cells that were administered differentiation medium.     

In Vitro Osteogenesis of Synovium Mesenchymal Cells Induced by Controlled Release of Alendronate and Dexamethasone from a Sintered Microspherical Scaffold

In vitro osteogenesis was successfully achieved with synovium-derived mesenchymal stem cells (SMSCs), which intrinsically have a strong chondrogenic tendency, by in situ release of alendronate (AL) and dexamethasone (Dex) from poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HA) sintered microspherical scaffold (PLGA/HA-SMS). Cumulative release profiles of AL and Dex from PLGA/HA-SMS and the influence on SMSCs osteogenic commitment were investigated. SMSCs seeded in Al-/Dex-loaded PLGA/HA-SMS (PLGA/HA-Com-SMS) exhibited significant osteogenic differentiation, as indicated by high yields of alkaline phosphatase (ALP) and bone calcification. In addition, mechanical properties (compressional) of PLGA/HA-Com-SMSs were also evaluated and approved. In conclusion, by promoting osteogenic commitment of SMSCs in vitro, this newly designed controlled-release system opens a new door to bone reparation and regeneration.     

Hydrostatic Pressure Enhances Chondrogenic Differentiation of Human Bone Marrow Stromal Cells in Osteochondrogenic Medium

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-β1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-β1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.     

The effect of PLGA doping of polycaprolactone films on the control of osteoblast adhesion and proliferation in vitro

Poly(epsilon-caprolactone) (PCL) film was modified using specified amounts of poly(d,l-lactide-co-glycolide) (PLGA) to provide a means to control polymer degradation. The aim of the study was to determine the effects of doping PCL with PLGA on the materials degradation, morphology and cell adhesion, to determine the significant variables within the process that could provide further control of cell adhesion. PLGA-doped PCL films were aged in osteogenic medium at 37 degrees C for up to 28 days. The aged samples were analysed in terms of weight loss or weight gain, molecule deposition and surface morphology. Molecule deposition was determined using Fourier transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) and morphology was determined using scanning electron microscopy and interferometric microscopy. The loss of the PLGA doping during degradation enhanced the formation of nano-porous structures in the remaining PCL domains, which attracted the deposition of substances from the osteogenic medium, which favoured the attachment and growth of human osteoblasts. The growth of osteoblasts was influenced by the controlled release of acidic products through polymer blending. Two pairs of FTIR-ATR absorption bands at 1090 and 1110 cm(-1), and at 1180 and 1190 cm(-1) were found to correlate to both PLGA and PCL, respectively. Changing the level of PLGA doping in PCL provided an approach to control the acidic products which can direct the growth of osteoblast cells.     

Effects of TGF-β3 and preculture period of osteogenic cells on the chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in a bilayered hydrogel composite

In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) and gelatin microparticles (MPs) were used to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-β3 (TGF-β3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-β3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-β3 significantly stimulated chondrogenic differentiation of MSCs. In addition, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-β3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-β3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites which mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.     

Esophageal epithelial cell interaction with synthetic and natural scaffolds for tissue engineering

As an initial step towards a tissue-engineered esophagus, rat esophageal epithelial cells (REEC) were isolated and characterized for epithelial identity, adhesion protein preference, and in vitro interaction with natural and synthetic scaffolds. The scaffolds consisted of AlloDerm (LifeCell Corporation, Branchburg, NJ), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic) acid (75:25) (PLGA75), poly(lactic-co-glycolic) acid (50:50) (PLGA50), and polycaprolactone/poly(L-lactic acid) (50:50) (PCL/PLLA). Various factors-including calcium concentration, scaffold composition, and pore size--were evaluated for their influence on epithelial growth and differentiation. By day 18, keratinocytes seeded on AlloDerm cultured under high Ca(++) (1.5mm) conditions showed a proliferating basal cell layer, epithelial stratification (5--6 layers) and a thick keratin layer. The synthetic scaffolds (PLGA, PLLA, PCL/PLLA) also showed complete surface coverage, regions of proliferating basal cells, and evidence of stratification (2--3 layers) and keratinization. The highly porous nature of the synthetic scaffolds, however, limited the formation of a continuous epithelial layer and resulted in a lack of overall spatially-defined differentiation. In conclusion, rat esophageal epithelial cells were successfully isolated and characterized, with cells seeded on AlloDerm showing superior epithelial organization and stratification compared to synthetic scaffolds. Modification of the synthetic scaffold's surface properties and pore size may be necessary to mimic epithelial behavior on natural scaffolds.     

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.     

In vitro generation of an osteochondral construct using injectable hydrogel composites encapsulating rabbit marrow mesenchymal stem cells

Injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized to fabricate a bilayered osteochondral construct consisting of a chondrogenic layer and an osteogenic layer, and to investigate the differentiation of rabbit marrow mesenchymal stem cells (MSCs) encapsulated in both layers in vitro. The results showed that MSCs in the chondrogenic layer were able to undergo chondrogenic differentiation, especially in the presence of TGF-β1-loaded MPs. In the osteogenic layer, cells maintained their osteoblastic phenotype. Although calcium deposition in the osteogenic layer was limited, cells in the osteogenic layer significantly enhanced chondrogenic differentiation of MSCs in the chondrogenic layer. The greatest effect was observed when MSCs were encapsulated with TGF-β1-loaded MPs and cultured with osteogenic cells in the bilayered constructs. Overall, this study demonstrates the fabrication of bilayered hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers.     

Bone augmentation by bone marrow mesenchymal stem cells cultured in three-dimensional biodegradable polymer scaffolds

Poly-lactic-glycolic acid (PLGA) is a biocompatible as well as biodegradable polymer and used in various medical applications. In this study, we evaluated efficiency of the specially designed three-dimensional porous PLGA as a scaffold for bone augmentation. First, cell attachment/proliferation, differentiation, and mineralization of Fisher 344 rat marrow mesenchymal stem cells (MSCs) cultured on the PLGA scaffold were analyzed. Viable MSCs were impregnated into pore areas of the scaffold and a moderate increase of DNA contents was seen. High alkaline phosphatase, osteocalcin content, and calcium content of MSCs in PLGA scaffolds under osteogenic differentiation conditions were seen after 14 or 21 days of culture. Subsequently, we implanted the PLGA/MSCs composites on rat calvaria bone for 30 days. Newly formed bone was seen in only the composite PLGA/MSCs implantation group, which had been precultured under osteogenic condition. We also demonstrated that the newly formed bone originated from the donor composites. These results demonstrate that the three-dimensional PLGA scaffold can support osteogenic differentiation of MSCs, and the scaffold combined with osteogenic MSCs can be used for in vivo bone tissue augmentation.     

Osteogenic induction of human periodontal ligament fibroblasts under two- and three-dimensional culture conditions

Human periodontal ligament fibroblasts (hPDLF) play a key role in the regeneration of periodontal compartment during guided tissue regeneration procedures. This property is attributed to the progenitor cell subsets residing in the area. The aim of this study was to investigate whether hPDLFs could undergo an osteogenic differentiation under two- and three-dimensional (2D and 3D) culture conditions upon osteogenic induction. hPDLFs were isolated from six healthy donors, cultured, and expanded according to standard protocols. Then, three osteogenic culture conditions (dexamethasone, ascorbic acid, and beta-glycerophosphate) were established: 1) 2D culture as single-cell monolayer, 2) 3D-static culture on mineralized poly(DL-lactic-co-glycolic acid) (PLGA) scaffold, and 3) 3D culture on mineralized PLGA scaffold inside the NASA-approved bioreactor stimulating microgravity conditions. After 21 days of osteogenic induction, the majority of monolayer cultures had undergone differentiation toward osteogenic lineage, as indicated by morphological changes, mineralization assay, and some phenotypical properties. However, immunohistochemistry revealed that the scaffold cultures expressed higher levels of osteogenic marker proteins compared with that of the monolayers. Secondly, hPDLF-PLGA constructs in bioreactor showed an increased expression of osteopontin and osteocalcin compared with that of static 3D culture after 21 days. Results indicate that human periodontal ligament contains a subpopulation of cells capable of undergoing osteogenic differentiation and presumably contributing to regeneration of bone defects in the adjacent area. Human PDLF-seeded mineralized PLGA scaffold in microgravity bioreactor may be used to support osteogenic differentiation in vitro. Thus, this system may offer new potential benefits as a tool for periodontal tissue engineering.     

The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering

Previous work on 2D synthetic films showed growth of human bladder stromal cells was enhanced on materials with lower moduli that mimic the elastic properties of native tissue. This study developed 3D synthetic foam scaffolds for soft tissue engineering by emulsion freeze-drying. Foams of poly(lactide-co-glycolide) (PLGA) and poly(?-caprolactone) (PCL) were extensively characterised using scanning electron microscopy, mercury porosimetry, dynamic mechanical analysis, degradation analysis, size exclusion chromatography and differential scanning calorimetry. Foams of 85–88% porosity and 35 μm pore diameter were selected for further study; the storage modulus of PCL foams was around half that of PLGA (2 MPa vs 4 MPa) and closer to the reported value for native bladder tissue. Urinary tract stromal cells showed a 4.4 and 2.4-fold higher attachment and rate of growth, respectively, on PCL scaffolds, as assessed by a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay. A greater contractile force was exerted by cells seeded in PLGA than on PCL scaffolds, raising the possibility that the reduced rate of proliferation of cells on PLGA scaffolds may reflect differentiation into a contractile phenotype. This study has generated PCL foam scaffolds with properties that may be pertinent to the tissue engineering of the bladder and other soft tissues.     

Characterization of osteoblast-like behavior of cultured bone marrow stromal cells on various polymer surfaces

The creation of novel bone substitutes requires a detailed understanding of the interaction between cells and materials. This study was designed to test certain polymers, specifically poly(caprolactone) (PCL), poly(D,L-lactic-CO-glycolic acid) (PLGA), and combinations of these polymers for their ability to support bone marrow stromal cell proliferation and differentiation. Bone marrow stromal cells were cultured from New Zealand White rabbits and were seeded onto glass slides coated with a thin layer of PCL, PLGA, and combinations of these two polymers in both a 40:60 and a 10:90 ratio. Growth curves were compared. At the end of 2 weeks, the cells were stained for both matrix mineralization and alkaline phosphatase activity. There was no statistically significant difference in growth rate of the cells on any polymer or polymer combination. However, there was a striking difference in Von Kossa staining and alkaline phosphatase staining. Cells on PCL did not show Von Kossa staining or alkaline phosphatase staining. However, in the 40:60 and 10:90 blends, there was both positive Von Kossa and alkaline phosphatase staining. These data indicate that PCL alone may not be a satisfactory material for the creation of a bone substitute. However, it may be used in combination with PLGA for the creation of a bone substitute material.     

小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

背景：研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9 的浓度呈剂量依赖正相关关系。 目的：通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原 mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。 方法：取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。 结果与结论：第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原 mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P > 0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。     

Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrospun PLGA nanofiber scaffold

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(d,l-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760+/-210 nm. The average Young's modulus of electrospun PLGA nanofibers was 42+/-26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1-4 weeks at a density of 2 x 10(6)cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells.     

Poly(epsilon-caprolactone) and poly (L-lactic-co-glycolic acid) degradable polymer sponges attenuate astrocyte response and lesion growth in acute traumatic brain injury

This study evaluated the response of rat brain to 2 degradable polymers (poly (L-lactic-co-glycolic acid) (PLGA), and poly(epsilon-caprolactone) (PCL)), two common materials in tissue engineering. PLGA has been extensively studied in the brain for controlled drug release as injectable microspheres and is generally accepted as biocompatible in that capacity. Biocompatibility in other forms and for different functions in the brain has not been widely studied. PCL was chosen as an alternative to PLGA for its slower degradation and less-acidic pH upon degradation. Porous scaffolds were made from both polymers and implanted into rat cerebral cortex for 1 and 4 weeks. Morphology, defect size, activation of microglia (OX-42) and astrocytes (glial fibrillary acidic protein (GFAP)), infiltration of activated macrophages (major histocompatibility complex (MHC)-II), and ingrowth of neurons (beta-tubulin type III (Tuj-1)) and progenitor cells (nestin) were analyzed using hematoxylin and eosin staining and immunofluorescence. PCL induced a lower inflammatory response than PLGA, as demonstrated by lower MHC-II and GFAP expression and greater ingrowth. Both polymers alleviated astrocytic activation and prevented enlargement of the defect. Tuj-1-, nestin-, and GFAP-positive cells were observed growing on both polymers at the peripheries of the sponge implants, demonstrating their permissiveness to neural ingrowth. These findings suggest that both polymers attenuate secondary death and scarring and that PCL might have advantages over PLGA.     

Multidifferentiation potential of mesenchymal stem cells in three-dimensional collagen gel cultures

Mesenchymal stem cells (MSCs) have a great therapeutic potential resulting from their ability to differentiate into multiple tissues when cultured under specific conditions. However, it has not been clearly demonstrated whether or not MSCs exhibit a multidifferentiation potential in three-dimensional collagen gel cultures. This study was conducted to explore the multidifferentiation potential of MSCs cultured in three-dimensional collagen gels. Human MSCs were cultured in 0.3% collagen gel for 20 days in chondrogenic differentiation medium (CDM), and for 14 days in osteogenic differentiation medium (ODM). Increases in GAG deposits, intensity of toluidine blue staining, and mRNA expressions of chondrogenic markers (type II collagen and type X collagen) were found in human MSCs cultured in the collagen gel maintained in CDM. Positive staining for alkaline phosphatase (ALP) activity and alizarin red, and increases in mRNA expressions of osteogenic markers (type I collagen, bone sialoprotein and ALP) were noted in the MSCs maintained in ODM. These findings emphasize that human MSCs have an ability to differentiate into both bone and cartilaginous tissues in three-dimensional collagen gel cultures, indicating potential clinical applications of MSC transplant therapy with collagen gel as a scaffold for bone or cartilage regeneration in complicated tissue defects.     

A novel approach for studying the temporal modulation of embryonic skeletal development using organotypic bone cultures and microcomputed tomography

Understanding the structural development of embryonic bone in a three dimensional framework is fundamental to developing new strategies for the recapitulation of bone tissue in latter life. We present an innovative combined approach of an organotypic embryonic femur culture model, microcomputed tomography (μCT) and immunohistochemistry to examine the development and modulation of the three dimensional structures of the developing embryonic femur. Isolated embryonic chick femurs were organotypic (air/liquid interface) cultured for 10 days in either basal, chondrogenic, or osteogenic supplemented culture conditions. The growth development and modulating effects of basal, chondrogenic, or osteogenic culture media of the embryonic chick femurs was investigated using μCT, immunohistochemistry, and histology. The growth and development of noncultured embryonic chick femur stages E10, E11, E12, E13, E15, and E17 were very closely correlated with increased morphometric indices of bone formation as determined by μCT. After 10 days in the organotpyic culture set up, the early aged femurs (E10 and E11) demonstrated a dramatic response to the chondrogenic or osteogenic culture conditions compared to the basal cultured femurs as determined by a change in μCT morphometric indices and modified expression of chondrogenic and osteogenic markers. Although the later aged femurs (E12 and E13) increased in size and structure after 10 days organotpypic culture, the effects of the osteogenic and chondrogenic organotypic cultures on these femurs were not significantly altered compared to basal conditions. We have demonstrated that the embryonic chick femur organotpyic culture model combined with the μCT and immunohistochemical analysis can provide an integral methodology for investigating the modulation of bone development in an ex vivo culture setting. Hence, these interdisciplinary techniques of μCT and whole organ bone cultures will enable us to delineate some of the temporal, structural developmental paradigms and modulation of bone tissue formation to underpin innovative skeletal regenerative technology for clinical therapeutic strategies in musculoskeletal trauma and diseases.     

Importance of integrin beta1-mediated cell adhesion on biodegradable polymers under serum depletion in mesenchymal stem cells and chondrocytes

To evaluate the predominant mechanism of chondrogenic cell [mesenchymal stem cells (MSCs) and chondrocytes] adhesion under serum free conditions, we measured the surface roughness and wettability of poly(lactic acid:polyglycolic acid=75:25) (PLGA), poly(lactic acid) (PLA), and poly(-epsilon-caprolactone) (PCL)-coated glass plates. Also to evaluate the biological reactions involved in cell-polymer interactions, integrin beta1, one of the cell adhesion molecules, was blocked with monoclonal antibody. In cell attachment test, MSCs and chondrocytes adhesion to synthetic polymers in 1h were very low and ranged from 2.8% to 8.0%. In present study, the correlation between attachment rate and surface roughness, contact angle, or integrin beta1 blocking on PLGA, PLA and PCL-coated plates could not be proved. However, we found that L-arginine-coated PLA highly increased the attachment rates of MSCs (30.2%) and of chondrocytes (26%), whereas integrin beta1 blocking significantly decreased these attachment rates to 5.6% and 7.4%, respectively, suggesting that increased cell adhesion to L-arginine-coated plates is mediated by integrin beta1.In this study, we showed that polymer characteristics such as roughness and wettability did not play an important role in cell adhesion under serum free conditions, because there was no significant difference according to polymer characteristics, whereas biological interactions mediated by integrin beta1 were critical during the early period of cell adhesion. The results suggest that L-arginine could be useful for facilitating early cell adhesion to synthetic polymers in cartilage tissue engineering.