Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.     

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.     

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

Collagen–PCL Sheath–Core Bicomponent Electrospun Scaffolds Increase Osteogenic Differentiation and Calcium Accretion of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hASCs) are an abundant cell source capable of osteogenic differentiation, and have been investigated as an autologous stem cell source for bone tissue engineering applications. The objective of this study was to determine if the addition of a type-I collagen sheath to the surface of poly(ε-caprolactone) (PCL) nanofibers would enhance viability, proliferation and osteogenesis of hASCs. This is the first study to examine the differentiation behavior of hASCs on collagen–PCL sheath–core bicomponent nanofiber scaffolds developed using a co-axial electrospinning technique. The use of a sheath–core configuration ensured a uniform coating of collagen on the PCL nanofibers. PCL nanofiber scaffolds prepared using a conventional electrospinning technique served as controls. hASCs were seeded at a density of 20 000 cells/cm² on 1 cm² electrospun nanofiber (pure PCL or collagen–PCL sheath–core) sheets. Confocal microscopy and hASC proliferation data confirmed the presence of viable cells after 2 weeks in culture on all scaffolds. Greater cell spreading occurred on bicomponent collagen–PCL scaffolds at earlier time points. hASCs were osteogenically differentiated by addition of soluble osteogenic inductive factors. Calcium quantification indicated cell-mediated calcium accretion was approx. 5-times higher on bicomponent collagen–PCL sheath–core scaffolds compared to PCL controls, indicating collagen–PCL bicomponent scaffolds promoted greater hASC osteogenesis after two weeks of culture in osteogenic medium. This is the first study to examine the effects of collagen–PCL sheath–core composite nanofibers on hASC viability, proliferation and osteogenesis. The sheath–core composite fibers significantly increased calcium accretion of hASCs, indicating that collagen–PCL sheath–core bicomponent structures have potential for bone tissue engineering applications using hASCs.     

Core-shell PLGA/collagen nanofibers loaded with recombinant FN/CDHs as bone tissue engineering scaffolds

Poly(lactic-co-glycolic acid) (PLGA)/collagen nanofibrous scaffolds have been utilized in the tissue engineering field. It has been shown that both fibronectin (FN) and cadherin 11 (CDH) play important roles in the progress of osteogenesis and cell adhesion. The aim of this study was to fabricate recombinant FN/CDHs (rFN/CDHs)-loaded PLGA/collagen nanofibrous scaffolds and evaluate their effects on the adhesion and differentiation of human bone marrow mesenchymal stem cells (hMSCs). PLGA/collagen nanofibers were made by coaxial electrospinning. The morphology and mechanical properties of PLGA/collagen nanofibrous mats were analyzed by scanning electron microscopy and mechanical testing, respectively. The performance of scaffolds was evaluated in terms of the viability, morphology, and osteogenic gene expression levels of hMSCs. rFN/CDHs was successfully incorporated into the PLGA/collagen nanofibers. The release of rFN/CDHs from PLGA nanofibers was investigated by liquid chromatography–mass spectrometry. rFN/CDHs improved the mechanical properties of the PLGA/collagen nanofibers. The controlled release of rFN/CDHs can enhance the proliferation of hMSCs and induce osteogenic gene expression (alkaline phosphatase, RUNX2, and osteocalcin). Our data imply that rFN/CDHs may induce hMSCs differentiation into osteoblasts and PLGA/collagen nanofibers loaded with rFN/CDHs have potential in bone tissue engineering.     

Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow

We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.     

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.     

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.     

Collagen-calcium phosphate cement scaffolds seeded with umbilical cord stem cells for bone tissue engineering

Human umbilical cord mesenchymal stem cells (hUCMSCs) avoid the invasive procedure required to harvest bone marrow MSCs. The addition of collagen fibers into self-setting calcium phosphate cement (CPC) may increase the scaffold strength, and enhance cell attachment and differentiation. The objectives of this study were to develop a novel class of collagen-CPC composite scaffolds, and to investigate hUCMSC attachment, proliferation, and osteogenic differentiation on collagen-CPC scaffolds for the first time. Collagen fibers in CPC improved the load-bearing capability. Flow cytometry showed that the hUCMSCs expressed cell surface markers characteristic of MSCs, and were negative for hematopoietic and endothelial cell markers. hUCMSCs proliferated rapidly in all CPC composite scaffolds, with cell number increasing by sevenfold in 8 days. Cellular function was enhanced with collagen fibers in CPC scaffolds. Cell density increased from (645±60) cells/mm(2) on CPC with 0% collagen, to (1056±65) cells/mm(2) on CPC with 8% collagen (p<0.05). The actin stress fibers inside the hUCMSCs were stained, and the fluorescence intensity was doubled when the collagen in CPC was increased by 0% to 8%. RT-PCR showed that hUCMSCs on CPC with collagen had higher osteogenic expression than those on CPC without collagen. Alizarin Red S staining revealed a great increase in mineralization by hUCMSCs on CPC with collagen than that without collagen. In conclusion, hUCMSCs showed excellent proliferation, differentiation, and synthesis of bone minerals in collagen-CPC composite scaffolds for the first time. The novel hUCMSC-seeded collagen-CPC construct with superior cell function and load-bearing capability is promising to enhance bone regeneration in a wide range of orthopedic and craniofacial applications.     

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2)

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.     

Collagen three-dimensional hydrogel matrix carrying basic fibroblast growth factor for the cultivation of mesenchymal stem cells and osteogenic differentiation

Three-dimensional (3D) collagen hydrogels have been extensively used for cell culture experiments and are more closely representative of in vivo conditions than monolayer (2D) culture. Here we cultured rat bone marrow-derived mesenchymal stem cells (MSCs) in collagen hydrogels containing varying concentrations of basic fibroblast growth factor (bFGF) to examine the effect of bFGF on MSC proliferation and osteogenic differentiation in 3D culture. The optimal bFGF concentration that promoted the greatest degree of cell proliferation and expression of the early osteogenic induction marker alkaline phosphatase was also determined. Subsequent quantitative real-time polymerase chain reaction analysis of gene expression demonstrated that bFGF promoted significant upregulation of the bone-related genes: collagen type I, osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) for periods of up to 21 days. Immunofluorescence staining and fluorescence-activated cell sorting analysis further supported the enhanced osteogenic differentiation of cells as a greater proportion of cells were found to express OPN. Matrix mineralization within the collagen hydrogels was enhanced in the presence of bFGF, as assessed by calcium detection using von Kossa staining. These results clearly demonstrate a positive effect of bFGF on proliferation and osteogenic induction of MSCs in 3D collagen hydrogels when applied at the appropriate concentration. Moreover, collagen hydrogel constructs containing MSCs and appropriate growth factor stimulus might be a potentially useful biological tool for 3D bone tissue engineering.     

Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion

Mesenchymal stem cells (MSCs) represent an attractive cell source for tissue engineering applications, since they are readily isolated from adult bone marrow and have the ability to differentiate along multiple mesenchymal lineages, including osteogenic. Currently, utilization of MSCs for bone tissue engineering is limited because of the attenuation of their osteogenic differentiation potential and in vivo bone-forming capacity following ex vivo expansion on conventional tissue culture plastic (TCP). Previously, we demonstrated that a denatured type I collagen (DC) matrix promotes the maintenance of MSC in vitro osteogenic differentiation potential during ex vivo expansion in contrast to TCP. In this study, we further demonstrate that the maintenance of MSC osteogenic differentiation potential is primarily due to the ability of DC matrix to influence the retention of early passage osteogenic functions in late passage (LP) cells during ex vivo expansion, in contrast to solely enhancing attenuated LP cellular functions during osteogenic differentiation. Serum-associated factors played a significant role in influencing the retention of MSC osteogenic differentiation potential during expansion on the DC matrix. Significantly, the results show that although LP cells expanded ex vivo on TCP highly attentuate their in vivo bone-forming capacity, the expansion of MSCs on DC matrix preserves this ability as determined by histological, histomorphometric, and bone mineral density evaluations of MSC-seeded hydroxyapatite/tricalcium phosphate scaffolds following an 8-week implantation period within a heterotopic muscle pouch model. These findings provide further insight into the importance of matrix-mediated effects on MSC function and selective factors important in this process.     

Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent

Macroporous composites made of coralline:chitosan with new microstructural features were studied for their scaffolding potential in in vitro bone regeneration. By using different ratios of natural coralline powder, as in situ gas forming agent and reinforcing phase, followed by freeze-drying, scaffolds with controlled porosity and pore structure were prepared and cultured with mesenchymal stem cells (MSCs). Their supportive activity of cellular attachment, proliferation and differentiation were assessed through cell morphology studies, DNA content, alkaline phosphatase (ALP) activity and osteocalcin (OC) release. The coralline scaffolds showed by far the highest evaluation of cell number and ALP activity over all the other chitosan-based scaffolds. They were the only material on which the OC protein was released throughout the study. When used as a component of the chitosan composite scaffolds, these coralline's favourable properties seemed to improve the overall performance of the chitosan. Distinct cell morphology and osteoblastic phenotype expression were observed depending on the coralline-to-chitosan ratios composing the scaffolds. The coralline-chitosan composite scaffolds containing high coralline ratios generally showed higher total cell number, ALP activity and OC protein expression comparing to chitosan scaffolds. The results of this study strongly suggest that coralline:chitosan composite, especially those having a high coralline content, may enhance adhesion, proliferation and osteogenic differentiation of MSCs in comparison with pure chitosan. Coralline:chitosan composites could therefore be used as attractive scaffolds for developing new strategies for in vitro tissue engineering.     

rhBMP-2／PLGA缓释支架的制备及对MSCs细胞成骨活性的体外研究

目的探讨骨形态发生蛋白（rhBMP-2）的聚乳酸聚乙醇酸共聚物（PLGA）体外缓释生物支架对人骨髓间充质干细胞（MSCs）细胞的影响。方法采用粒子沥滤-冷冻干燥复合工艺制备了附载rhBMP-2的PLGA生物支架,并检测了在PLGA的降解过程中rhBMP-2的释药规律;同时分离培养人骨髓间充质干细胞,体外培养后分别接种于附载和未附载rhBMP-2的PLGA支架上。扫描电镜观察不同时间段MSC在支架上的生长情况;MTT法测定细胞增殖情况。结果rhBMP-2能被包裹进PLGA支架中,而且可以在PLGA支架降解过程中持续释放出来并诱导骨发生。结论骨形态发生蛋白的PLGA复合载体是一种较为理想的新型生物支架。     

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.     

A comparison of the osteogenic potential of adult rat mesenchymal stem cells cultured in 2-D and on 3-D collagen glycosaminoglycan scaffolds.

Adult mesenchymal stem cells (MSCs) have the capability to differentiate along several lineages including those of bone, cartilage, tendon and muscle, thus offering huge potential for the field of tissue engineering. The purpose of this study was to characterise the differentiation capacity of rat MSCs cultured on standard plastic coverslips in 2 dimensions and on a novel collagen glycosaminoglycan scaffold in the presence of a standard combination of osteoinductive factors. Cells were cultured for 3, 7, 14 and 21 days and several markers of osteogenesis were analysed. While the initial response of the cells in 3-D seemed to be faster than cells cultured in 2-D, as evidenced by collagen type I expression, later markers showed that osteogenic differentiation of MSCs took longer in the 3-D environment of the collagen GAG scaffold compared to standard 2-D culture conditions. Furthermore, it was shown that complete scaffold mineralisation could be evoked within a 6 week timeframe. This study further demonstrates the potential use of MSC-seeded collagen GAG scaffolds for bone tissue engineering applications.     

Comparative study of osteogenic differentiation potential of mesenchymal stem cells derived from bone marrow and adipose tissue of osteoporotic female rats

Osteoporosis causes reduction of osteogenic differentiation of mesenchymal stem cells (MSCs) from bone marrow and adipose tissue. This study was designed to compare the osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) and adipose-derived stem cells (ADSCs) of ovariectomized (OVX) rats. MSC were harvested from bone marrow and inguinal fat pads of six OVX rats. The limitations of this report are that cells from different animals were pooled for the purpose of the experiments that were carried out in this study. At 7, 14 and 21?d of osteogenic differentiation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity and gene expression for collagen I, osteocalcin, bone sialoprotein, osteopontin and bone morphogenetic protein-2 bone morphogenetic protein-2 (BMP-2) were analyzed. At 21?d, percentage of cells per field and percentage of mineralized nodule were analyzed. The data were subjected to analysis of variance, and the means were compared by Student–Newman–Keuls test. The cells, regardless of group, showed phenotypic characteristics consistent with stem cells. MTT conversion, alkaline phosphatase activity, percentage of mineralized nodule and expression of collagen I, osteocalcin and BMP-2 of ADSCs from OVX rats were higher when compared to BMMSCs from OVX rats in at least one of the evaluated periods (p?p?相似文献   

Development of custom-built bone scaffolds using mesenchymal stem cells and apatite-wollastonite glass-ceramics

There is a clinical need for new bone replacement materials that combine long implant life with complete integration and appropriate mechanical properties. We have used human mesenchymal stem cells (MSCs) to populate porous apatite-wollastonite (A-W) glass-ceramic scaffolds produced by the layer manufacturing technique, selective laser sintering, to create custom-built bone replacements. Confocal and scanning electron microscopy were used to determine optimal seeding densities and to demonstrate that MSCs adhered and retained viability on the surface of A-W scaffolds over a culture period of 21 days. We found a significant increase in the number of MSCs growing on the scaffolds over 7 days. Using bromodeoxyuridine incorporation we demonstrated that MSCs proliferated on the scaffolds. Using real-time PCR we analyzed the expression of the osteogenic markers alkaline phosphatase, collagen type-I, Cbfa-1, osteocalcin, osteonectin, and osteopontin by MSCs cultured in the absence of osteogenic supplements. The expression of the osteogenic markers by MSCs was equivalent to or significantly greater on A-W scaffolds than on tissue culture plastic. We also identified significantly higher alkaline phosphatase activity on A-W compared to a commercial calcium phosphate scaffold. These results indicate for the first time the biocompatibility and osteo-supportive capacity of A-W scaffolds and their potential as patient-specific bone replacement materials.     

Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering

This study reports the preparation of novel porous scaffolds of calcium phosphate cement (CPC) combined with alginate, and their potential usefulness as a three-dimensional (3-D) matrix for drug delivery and tissue engineering of bone. An α-tricalcium phosphate-based powder was mixed with sodium alginate solution and then directly injected into a fibrous structure in a Ca-containing bath. A rapid hardening reaction of the alginate with Ca(2+) helps to shape the composite into a fibrous form with diameters of hundreds of micrometers, and subsequent pressing in a mold allows the formation of 3-D porous scaffolds with different porosity levels. After transformation of the CPC into a calcium-deficient hydroxyapatite phase in simulated biological fluid the scaffold was shown to retain its mechanical stability. During the process biological proteins, such as bovine serum albumin and lysozyme, used as model proteins, were observed to be effectively loaded onto and released from the scaffolds for up to more than a month, proving the efficacy of the scaffolds as a drug delivering matrix. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and then cultured on the CPC-alginate porous scaffolds to investigate the ability to support proliferation of cells and their subsequent differentiation along the osteogenic lineage. It was shown that MSCs increasingly actively populated and also permeated into the porous network with time of culture. In particular, cells cultured within a scaffold with a relatively high porosity level showed favorable proliferation and osteogenic differentiation. An in vivo pilot study of the CPC-alginate porous scaffolds after implantation into the rat calvarium for 6 weeks revealed the formation of new bone tissue within the scaffold, closing the defect almost completely. Based on these results, the newly developed CPC-alginate porous scaffolds could be potentially useful as a 3-D matrix for drug delivery and tissue engineering of bone.