Fabrication and characterization of bioactive wollastonite/PHBV composite scaffolds

Composite scaffolds of polyhydroxybutyrate-polyhydroxyvalerate (PHBV) with bioactive wollastonite were fabricated by a compression moulding, thermal processing, and salt particulate leaching method. Structure and mechanical properties of the scaffolds were determined. The bioactivity of the composites was evaluated by soaking in a simulated body fluid (SBF), and the formation of the hydroxyapatite (HAp) layer was determined by Scanning Electron Microscope (SEM) and Energy-Dispersive Spectrometer (EDS). The results showed that the wollastonite/PHBV composites were bioactive as it induced the formation of HAp on the surface of the composite scaffolds after soaking in SBF for 14 days. In addition, the measurements of the water contact angles suggested that incorporation of wollastonite into PHBV could improve the hydrophilicity of the composites and the enhancement was dependent on the wollastonite content. Furthermore, the pH and ion concentration changes of SBF solutions with composite scaffolds showed that the composites released Ca and Si ions, which could neutralize the acidic by-products of the PHBV and stabilize the pH of the SBF solutions between 7.2 and 7.8 within a 3-week soaking period. All of these results suggest that the incorporation of wollastonite was a useful approach to obtain composite scaffolds with improved properties.     

Freeform fabricated scaffolds with roughened struts that enhance both stem cell proliferation and differentiation by controlling cell shape

We demonstrate that freeform fabricated (FFF) scaffolds with a roughened surface topography can support hBMSC proliferation, while also inducing osteogenic differentiation, for maximized generation of calcified, bone-like tissue. Previously, hBMSCs rapidly proliferated, without osteogenic differentiation, during culture in FFF scaffolds. In contrast, hBMSCs underwent osteogenic differentiation, with slow proliferation, during culture in nanofiber scaffolds. Analysis of cell morphology showed that the topography presented by the nanofiber scaffolds drove hBMSC differentiation by guiding them into a morphology that induced osteogenic differentiation. Herein, we hypothesized that using the high-surface area architecture of FFF scaffolds to present a surface roughness that drives hBMSCs into a morphology that induces osteogenic differentiation would yield a maximum amount differentiated hBMSCs and bone-like tissue. Thus, a solvent etching method was developed that imparted a 5-fold increase in roughness to the surface of the struts of poly(ε-caprolactone) (PCL) FFF scaffolds. The etched scaffolds induced osteogenic differentiation of the hBMSCs while un-etched scaffolds did not. The etched scaffolds also supported the same high levels of hBMSC proliferation that un-etched scaffolds supported. Finally, hBMSCs on un-etched scaffolds had a large spread area, while hBMSCs on etched scaffolds has a smaller area and were more rounded, indicating that the surface roughness from the etched scaffolds dictated the morphology of the hBMSCs. The results demonstrate that FFF scaffolds with surface roughness can support hBMSC proliferation, while also inducing osteogenic differentiation, to maximize generation of calcified tissue. This work validates a rational approach to scaffold fabrication where the structure of the scaffold was designed to optimize stem cell function by controlling cell morphology.     

Plasma sprayed wollastonite/TiO2 composite coatings on titanium alloys

Wollastonite/TiO2 composite coatings were prepared using plasma spraying technology onto Ti-6Al-4V substrate. The composite coatings exhibit obvious lamellar structure with alternating wollastonite coating and TiO2 coating. No obvious cracks exist on the interface between coatings and substrate. In the case of composite coatings, the primarily crystalline phases of the coatings are wollastonite and rutile, indicating wollastonite and TiO2 did not react during plasma spraying process. Some of rutile in the powders transforms into anatase due to plasma spraying. The mean bond strength of the composite coatings is higher than 30 MPa. The Vickers microhardness of coatings increase with the increase in the content of TiO2. Wollastonite/TiO2 composite coatings were soaked in simulated body fluid to examine their bioactivity. Carbonate-containing hydroxyapatite (CHA) layer was formed on the surface of the wollastonite and W7T3 coatings soaked in simulated body fluid, while was not formed on the surface of the TiO2 and W3T7 coatings after immersion. In addition, a rich-silica layer appeared at the interface of CHA and wollastonite and W7T3 coatings. In order to investigate the cytocompatibility of the coatings, osteoblast was seeded onto the surface of the coatings. The scanning electron microscopy observation showed that the addition of wollastonite promote the proliferation of osteoblast. It is enough to prove that the wollastonite and wollastonite/TiO2 composite coatings possess excellent cytocompatibility.     

Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold

The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable β-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.     

Biocompatibility assessment of polytetrafluoroethylene/wollastonite composites using endothelial cells and macrophages

The aim of the study was to prepare a composite of polytetrafluoroethylene/wollastonite (PTFE/W) and evaluate its biocompatibility with endothelial cells. A composite of PTFE with wollastonite in the proportion 90/10 w/w was prepared. The dynamic storage modulus of composite is found to increase from 260 to about 453 MPa at room temperature while a marginal increase is observed in the compressive modulus. Higher values of storage modulus of PTFE/W relative to pristine PTFE over a range of temperature indicated the contribution of wollastonite in improving the rigidity of PTFE. Electron microscopic visualization of composite surface indicates suitable morphology for cell growth with the cross-section showing no evidence of bonding between PTFE and wollastonite. The water contact angle of the composite indicates increased hydrophilicity over native PTFE due to the presence of wollastonite. A direct-contact test did not show any deleterious effects on endothelial cell morphology and viability, indicating its compatibility. Leached-out products (LOP) from the composite were determined to be non-toxic as tested by tetrazolium (MTT) and Neutral red uptake (NRU) assays. Mouse peritoneal macrophages cultured in the presence of the composites did not show upregulation of activation markers such as CD11b/CD 18 (Mac-1), CD45, CD 14, and CD86 (B7.2) in comparison to macrophages cultured in contact with PTFE alone, indicating its non-activating nature. LOP did not induce proliferation of mouse splenic lymphocytes suggesting its immuno-tolerance. In static incubation assay contact with composite did not lead to hemolysis thus exhibiting preliminary hemocompatibility of the material. Suitable physico-chemical properties and well tolerance by endothelial cells and macrophages make this composite a prospective biomaterial. One could foresee the applications of this composite in areas where materials need to possess high rigidity and are subject to elevated temperatures.     

Biocompatibility assessment of polytetrafluoroethylene/wollastonite composites using endothelial cells and macrophages

The aim of the study was to prepare a composite of polytetrafluoroethylene/wollastonite (PTFE/W) and evaluate its biocompatibility with endothelial cells. A composite of PTFE with wollastonite in the proportion 90/10 w/w was prepared. The dynamic storage modulus of composite is found to increase from 260 to about 453 MPa at room temperature while a marginal increase is observed in the compressive modulus. Higher values of storage modulus of PTFE/W relative to pristine PTFE over a range of temperature indicated the contribution of wollastonite in improving the rigidity of PTFE. Electron microscopic visualization of composite surface indicates suitable morphology for cell growth with the cross-section showing no evidence of bonding between PTFE and wollastonite. The water contact angle of the composite indicates increased hydrophilicity over native PTFE due to the presence of wollastonite. A direct-contact test did not show any deleterious effects on endothelial cell morphology and viability, indicating its compatibility. Leached-out products (LOP) from the composite were determined to be non-toxic as tested by tetrazolium (MTT) and Neutral red uptake (NRU) assays. Mouse peritoneal macrophages cultured in the presence of the composites did not show upregulation of activation markers such as CD11b/CD18 (Mac-1), CD45, CD14, and CD86 (B7.2) in comparison to macrophages cultured in contact with PTFE alone, indicating its nonactivating nature. LOP did not induce proliferation of mouse splenic lymphocytes suggesting its immuno-tolerance. In static incubation assay contact with composite did not lead to hemolysis thus exhibiting preliminary hemocompatibility of the material. Suitable physico-chemical properties and well tolerance by endothelial cells and macrophages make this composite a prospective biomaterial. One could foresee the applications of this composite in areas where materials need to possess high rigidity and are subject to elevated temperatures.     

Human urine-derived stem cells can be induced into osteogenic lineage by silicate bioceramics via activation of the Wnt/β-catenin signaling pathway

Human urine-derived stem cells (USCs) have great application potential for cytotherapy as they can be obtained by non-invasive and simple methods. Silicate bioceramics, including calcium silicate (CS), can stimulate osteogenic differentiation of stem cells. However, the effects of silicate bioceramics on osteogenic differentiation of USCs have not been reported. In this study, at first, we investigated the effects of CS ion extracts on proliferation and osteogenic differentiation of USCs, as well as the related mechanism. CS particles were incorporated into poly (lactic-co-glycolic acid) (PLGA) to obtain PLGA/CS composite scaffolds. USCs were then seeded onto these scaffolds, which were subsequently transplanted into nude mice to analyze the osteogenic differentiation of USCs and mineralization of extracellular matrix formed by USCs in vivo. The results showed that CS ion extracts significantly enhanced cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and expression of certain osteoblast-related genes and proteins. In addition, cardamonin, a Wnt/β-catenin signaling inhibitor, reduced the stimulatory effects of CS ion extracts on osteogenic differentiation of USCs, indicating that the observed osteogenic differentiation of USCs induced by CS ion extracts involves Wnt/β-catenin signaling pathway. Furthermore, histological analysis showed that PLGA/CS composite scaffolds significantly enhanced the osteogenic differentiation of USCs in vivo. Taken together, these results suggest the therapeutic potential of combining USCs and PLGA/CS scaffolds in bone tissue regeneration.     

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.     

In vitro osteogenic potential of human bone marrow stromal cells cultivated in porous scaffolds from mineralized collagen

Porous 3D structures from mineralized collagen were fabricated applying a procedure in which collagen fibril reassembly and precipitation of nanocrystalline hydroxyapatite (HA) occur simultaneously. The resulting matrices were evaluated in vitro with respect to their suitability as scaffolds for bone tissue engineering. We found a high capacity of the material to bind serum proteins as well as to absorb Ca2+ ions, which could be advantageous to promote cell attachment, growth, and differentiation. Human bone marrow stromal cells (hBMSCs) were seeded onto the 3D scaffolds and cultivated for 4 weeks in the presence and absence of osteogenic supplements. We studied viability, proliferation, and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content, and alkaline phosphatase (ALP) activity. Furthermore, the expression for bone-related genes (ALP, bone sialo protein II (BSP II), and osteocalcin) was analyzed. In our investigation we found a 2.5-fold to 5-fold raise in DNA content and an increase of ALP activity for osteogenic induced hBMSC on collagen HA scaffolds. The expression of ALP and BSP II in these cells was also stimulated in the course of cultivation; however, we did not detect an upregulation of osteocalcin gene expression. These data suggest, that porous collagen HA scaffolds are suitable for the expansion and osteogenic differentiation of hBMSC and are therefore promising candidates for application as bone grafts.     

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.     

Fabrication, characterization, and in vitro degradation of composite scaffolds based on PHBV and bioactive glass

Composite scaffolds of polyhydroxybutyrate-polyhydroxyvalerate (PHBV) with sol-gel-derived bioactive glass (BG, 58S) are fabricated by compression molding, thermal processing, and salt particulate leaching method. Structure and mechanical properties of the scaffolds are determined. The bioactivity of the composites is evaluated by soaking the scaffolds in a simulated body fluid (SBF), and the formation of the apatite layer on the scaffolds is determined by scanning electron microscopy (SEM) and energy-dispersive spectrometry (EDS). The results show that the PHBV/BG composites are bioactive as they induce the formation of apatite on the composite scaffolds after soaking in SBF for 3 days. In addition, the measurements of the water contact angles suggest that incorporation of BG into PHBV can improve the hydrophilicity of the composites and the enhancement is dependent on the BG content. Furthermore, the degradation assessment of the scaffolds is performed in phosphate-buffered saline (PBS) solution at 37 C. Weight loss and water absorption of the scaffolds, pH of the incubation media, and molecular weight measurements of the PHBV in the scaffolds are used to monitor the degradation of the scaffolds during a nine-week incubation in PBS. It has been found that the incorporation of bioactive glass into the PHBV delayed the degradation of PHBV in the composite scaffolds for the period investigated. The present results show not only a useful method to prepare composite scaffolds with improved properties but also a way of adjusting the in vitro degradation behavior of composite scaffolds by tailoring the content of bioactive glass.     

Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide

Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.     

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-β and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation.     

Modulus-driven differentiation of marrow stromal cells in 3D scaffolds that is independent of myosin-based cytoskeletal tension

Proliferation and differentiation of cells are known to be influenced by the physical properties of the extracellular environment. Previous studies examining biophysics underlying cell response to matrix stiffness utilized a two-dimensional (2D) culture format, which is not representative of the three-dimensional (3D) tissue environment in vivo. We report on the effect of 3D matrix modulus on human bone marrow stromal cell (hBMSC) differentiation. hBMSCs underwent osteogenic differentiation in poly(ethylene glycol) hydrogels of all modulus (300-fold modulus range, from 0.2?kPa to 59?kPa) in the absence of osteogenic differentiation supplements. This osteogenic differentiation was modulus-dependent and was enhanced in stiffer gels. Osteogenesis in these matrices required integrin-protein ligation since osteogenesis was inhibited by soluble Arginine-Glycine-Aspartate-Serine peptide, which blocks integrin receptors. Immunostained images revealed lack of well-defined actin filaments and microtubules in the encapsulated cells. Disruption of mechanosensing elements downstream of integrin binding that have been identified from 2D culture such as actin filaments, myosin II contraction, and RhoA kinase did not abrogate hBMSC material-driven osteogenic differentiation in 3D. These data show that increased hydrogel modulus enhanced osteogenic differentiation of hBMSCs in 3D scaffolds but that hBMSCs did not use the same mechanosensing pathways that have been identified in 2D culture.     

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.     

The determination of stem cell fate by 3D scaffold structures through the control of cell shape

Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage.     

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.     

Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering

Bionanocomposites formed by combining biodegradable polymers and nanosized osteoconductive inorganic solids have been regarded as promising biomimetic systems which possess much improved structural and functional properties for bone tissue regeneration. In this study three-dimensional nanocomposite scaffolds based on calcium phosphate (Ca-P)/poly(hydroxybutyrate–co-hydroxyvalerate) (PHBV) and carbonated hydroxyapatite (CHAp)/poly(l-lactic acid) (PLLA) nanocomposite microspheres were successfully fabricated using selective laser sintering, which is a rapid prototyping technology. The sintered scaffolds had controlled material microstructure, totally interconnected porous structure and high porosity. The morphology and mechanical properties of Ca-P/PHBV and CHAp/PLLA nanocomposite scaffolds as well as PHBV and PLLA polymer scaffolds were studied. In vitro biological evaluation showed that SaOS-2 cells had high cell viability and normal morphology and phenotype after 3 and 7 days culture on all scaffolds. The incorporation of Ca-P nanoparticles significantly improved cell proliferation and alkaline phosphatase activity for Ca-P/PHBV scaffolds, whereas CHAp/PLLA nanocomposite scaffolds exhibited a similar level of cell response compared with PLLA polymer scaffolds. The nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue engineering applications.     

Enhanced Cellular Responses of Human Bone Marrow Stromal Cells Cultured on Pretreated Surface with Allogenic Platelet-Rich Plasma

The principal objective of this study was to evaluate the effects of surface pretreatment with platelet-rich plasma (PRP) on the cellular functions of human bone marrow stromal cells (hBMSCs). The surfaces of tissue culture plates (TCPs) were pretreated by adding PRP followed by centrifugation to bring platelets closer to the surface, followed by incubation for 60 min at 37°C. Then, hBMSCs were seeded onto TCP and TCP pretreated with PRP (TCP-PRP), followed by culture in osteogenic medium. Cell attachment, proliferation, and osteogenic differentiation were evaluated. Field emission scanning electron microscope (FE-SEM; JSM-7401F, JEOL Ltd., Japan) observations were conducted. The attachment of hBMSCs was significantly lower on TCP-PRP than on TCP. However, when the cell numbers were normalized with those observed on day 1 of culture, cellular proliferation on 5 days was significantly higher on TCP-PRP. Alkaline phosphatase activity, an index of early phase of osteoblastic differentiation, was significantly higher on TCP-PRP on day 14. Calcium deposition amount, an index of terminal osteoblastic differentiation, was also significantly higher on TCP-PRP on days 14 and 21. The results of von Kossa staining confirmed that, on day 21, the area of mineralized nodules was significantly larger on TCP-PRP. FE-SEM observation demonstrated that activated platelets and fibrin network covered the surface after PRP treatment. An increase in the number of hBMSCs and their cellular products was evident on the FE-SEM observation, and the fibrin network remained on day 21. Our results demonstrate that a PRP-treated surface enhanced early proliferation and late osteogenic differentiation of hBMSCs.