Molded porous poly (L-lactide) membranes for guided bone regeneration with enhanced effects by controlled growth factor release

The aim of this study was to develop platelet-derived growth factor (PDGF-BB) loaded moldable porous poly (L-lactide) (PLLA)-tricalcium phosphate (TCP) membranes for guided bone regeneration (GBR) therapy. The membranes were designed to fit various types of bone defect sites. PDGF-BB-dissolved PLLA-TCP in methylene chloride-ethyl acetate solution was cast on a dome shaped metallic mold to fabricate a model membrane. The release rate of PDGF-BB, the osteoblast attachment test, and guided bone regeneration potential were evaluated with PDGF-BB-loaded PLLA-TCP membranes. Regular pores were generated throughout the membrane mainly due to phase inversion of PLLA-methylene chloride-ethyl acetate solution. A therapeutic amount of PDGF-BB was released from the membrane. The release rate could be controlled by varying the initial loading content of PDGF-BB. A significant amount of cells attached onto the PDGF-BB-loaded membrane rather than onto the unloaded membrane. Dome shaped bone formation was achieved in rabbit calvaria at 4 weeks. This indicated that restoration of bone defects to the bone's original shape can be made possible by using molded membranes, which guide bone regeneration along with providing sufficient spaces. Bone forming efficiency was increased remarkably due to PDGF-BB release from PLLA-TCP membranes. These results suggested that the PDGF-BB releasing molded PLLA-TCP membrane may potentially improve GBR efficiency in various types of bone defects.     

Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.     

Natural marine sponge fiber skeleton: a biomimetic scaffold for human osteoprogenitor cell attachment, growth, and differentiation

Identification of suitable scaffolds onto which human stem cells can be seeded to generate functional three-dimensional tissues is a major research goal. A natural marine sponge skeleton was selected as a potential scaffold on the basis of the hydration potential of the fiber, the presence of open interconnected channels created by the fiber network, the collagenous composition of the fiber, and the structural diversity of fiber architecture. The skeleton of an undetermined species of Spongia (Class Demospongiae: Order Dictyoceratida: Family Spongiidae), composed of spongin, supported growth of human osteoprogenitor cells. Cell attachment and invasion into the framework were observed within 16 h, followed by development into membranous sheets between the sponge fibers by concentric infilling. Histochemical staining for alkaline phosphatase and type I collagen indicated formation of bone matrix as confirmed by birefringence. At 9 and 14 days alkaline phosphatase-specific activity in sponge fiber-osteoprogenitor cell cultures was significantly greater than in control cultures on cell culture plastic. Adsorption with recombinant human bone morphogenetic protein 2 confirmed the potential of this sponge skeleton as a delivery scaffold for osteogenic factors. The abundance and structural diversity of natural marine sponge skeletons and their potential as multifunctional, cell conductive and inductive frameworks indicate a promising new source of scaffold for tissue regeneration.     

The pro-angiogenic characteristics of a cross-linked gelatin matrix

To overcome limitations on regeneration in the nervous system and other organs caused by insufficient blood supply, we have developed a gelatin sponge material which stimulates blood vessel formation, i.e. angiogenesis. Controlled chemical cross-linking was employed to slow down enzymatic degradation of the gelatin matrix. Four different in vitro assays using L929 fibroblasts and purified endothelial cells indicated that the sponge material did not release toxic components, but provided a permissive substratum for cell attachment, cell migration and pronounced cell proliferation, all of which are crucial for the formation of vasculature. Two in vivo models were employed to directly monitor the pro-angiogenic impact of the sponge material. Implantation of gelatin sponges onto the chorioallantoic membrane of fertilized chicken eggs induced robust attraction of endothelial cells and formation of blood vessels. Angiogenesis inside gelatin implants occurred more than 200 times faster than in a commercial collagen sponge. Similarly, after subcutaneous implantation of tube-like sponges into mice, an increasing immigration of cells and subsequent formation of functional vasculature became evident. Immunocytochemistry revealed no fibronection accumulation and no scarring. In summary, our matrix based on cross-linked gelatin promises to be a valuable component of future implants, improving neuronal and non-neuronal regeneration by concomitant pro-angiogenic stimulation.     

Proliferation and differentiation of rat bone marrow stromal cells on poly(glycolic acid)-collagen sponge

We studied the effects of dexamethasone (Dex) and basic fibroblast growth factor (bFGF) on proliferation and differentiation of rat bone marrow stromal cells (RBMSCs), using three scaffolds: collagen sponge, poly(glycolic acid) (PGA)-collagen sponge, and PGA-collagen (UV) sponge. RBMSCs were seeded into the sponges, and cultured in primary medium, primary medium with Dex, and primary medium with bFGF and Dex. Three weeks after cultivation, we examined alkaline phosphatase (ALP) activity and cell number in the sponges, and also performed macroscopic, light microscopic, and scanning electron microscopic (SEM) observations. Collagen sponge shrank considerably, but PGA-collagen and PGA-collagen (UV) sponges maintained most of their original shape. PGA-collagen (UV) sponge supplemented with bFGF and Dex together had the highest ALP activity and cell number, followed by PGA-collagen sponge. Although collagen sponge showed cell proliferation only on the surface, the other two sponges showed cell proliferation in the interior. SEM showed the best cell attachment to PGA-collagen (UV) sponge in the presence of bFGF and Dex, followed by PGA-collagen sponge. In conclusion, PGA-collagen (UV) and PGA-collagen sponges proved to be much more useful as scaffolding for bone regeneration when combined with bFGF and Dex.     

Enhanced proliferation and osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with different poly(ethylene terephthalate) fibers

The objective of this study was to investigate the feasibility of collagen sponges mechanically reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers in stem cell culture. A collagen solution with homogeneously dispersed PET fibers was freeze-dried, followed by dehydrothermal cross-linking to obtain the collagen sponge incorporating PET fibers. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 200 microm, irrespective of PET fiber incorporation. As expected, PET fibers incorporation significantly enhanced the compression strength of collagen sponge. When used for rat mesenchymal stem cells (MSC), the collagen sponge incorporating PET fibers was superior to the original collagen sponge without PET fibers incorporation in terms of the initial attachment, proliferation and osteogenic differentiation of cells, irrespective of the amount and diameter of fibers incorporated. The shrinkage of sponges during cell culture was significantly suppressed by the fiber incorporation. It is possible that the shrinkage suppression maintains the three-dimensional inner pore structure of collagen sponges without impairing the cell compatibility, resulting in the superior MSC attachment and the subsequent osteogenic differentiation in the sponge incorporating PET fiber.     

联合应用血管内皮生长因子及血小板衍生生长因子BB干预骨髓间充质干细胞的血管化及增殖能力

背景：组织工程骨为修复极限骨缺损提供了新的选择,但只有先形成完善的功能性血管网,保证稳定的成骨和骨整合,才能取得良好的治疗效果。因此,血管化可以说是组织工程骨面临的最大挑战与难题。 目的：探讨体外联合应用血管内皮生长因子(vascular endothelial growth factor,VEGF)和血小板衍生生长因子BB(platelet-derived growth factor-BB,PDGF-BB)对骨髓间充质干细胞增殖和成血管能力的影响。 方法：体外分离培养SD大鼠骨髓间充质干细胞,分别用不同质量浓度VEGF(20,40,60,80,100,120 μg/L)、PDGF-BB(20,40,60,80,100,120 μg/L)联合干预以及100 μg/L VEGF单独干预、100 μg/L PDGF-BB单独干预,CCK-8实验检测2种细胞因子促进细胞增殖的最佳质量浓度,然后在第7天和第14天通过RT-PCR方法检测血管生成素1、缺氧诱导因子1α、肝细胞生长因子、胰岛素样生长因子等相关成血管基因的表达量。 结果与结论：①加入生长因子后,细胞增殖能力明显提高,联合作用效果更优,最佳组合为80 μg/L VEGF+   80 μg/L PDGF-BB;②VEGF、PDGF-BB都可以促进血管生成素1、缺氧诱导因子1α、肝细胞生长因子和胰岛素样生长因子的mRNA表达,联合应用时效果最佳;③缺氧诱导因子1α、肝细胞生长因子 mRNA表达随时间延长有所升高,差异有显著性意义(P < 0.05);而血管生成素1、胰岛素样生长因子 mRNA表达量随时间延长有所降低,差异有显著性意义(P < 0.05);④体外实验结果证明,当VEGF和PDGF-BB质量浓度均为80 μg/L时,能够持续稳定促进整个血管形成过程,且促进作用优于单独一种生长因子。ORCID: 0000-0003-1918-579X(何惠宇) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Heat-treated membranes with bioelectricity promote bone regeneration

The barrier membranes maintain a secluded space to prevent the ingrowth of connective tissue and direct the growth of new bone into a desired site; however, they do not stimulate or induce bone regeneration. To enhance the bone bioactivities of membranes, we developed chitosan electret membranes with bioelectricity by grid-controlled constant voltage corona charging. The electret membranes charged with heat treatment (HT electret membranes) exhibited superior electret charge storage stability than the ones charged without heat treatment (RT electret membranes). Human bone marrow stromal cells (hBMSCs) demonstrated better growth on HT electrets membrane. Moreover, hBMSCs osteoblastic differentiation was enhanced on HT electret membranes, as evidenced by osteocalcin and osteopontin expression as assessed by immunocytochemistry, quantitative RT-PCR and western blot analysis. The rabbit calvarial defect model demonstrated that HT electret membranes induced a significantly enhanced bone regeneration compared with RT electret membranes. New bone formation was found at both the periphery and in the center of the defects four weeks after implantation. These results indicated that the chitosan electret membrane has osteogenic potential and could be applied as a novel barrier membrane.     

Bioluminescent and micro-computed tomography imaging of bone repair induced by fibrin-binding growth factors

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.     

Peripheral nerve regeneration through the space formed by a chitosan gel sponge

The clinical treatment of traumatized peripheral nerves often requires grafting of autologous cutaneous nerves. However, there are drawbacks in sacrificing healthy nerves and tissue scarring. In this study, an artificial material, freeze-dried chitosan gel sponge, was examined as a scaffold for nerve regeneration in rats. An 8-mm gap was made by removing a segment of the sciatic nerve, and the distal and proximal stumps were sandwiched by chitosan gel sponge. Rats were killed at 4, 7, 14, and 28 days, and 2 and 4 months after the operation and histological and morphometric evaluations were performed. Regenerating axons were observed at 4 days after the operation. Regenerating nerves extended the distal stump at 14 days after surgery. By electron microscopy, numerous macrophages appeared to phagocyte chitosan, and made a dense cell layer on the chitosan. Regenerating axons did not touch the chitosan, and extended through the space surrounded by macrophage-stacked chitosan. Regenerating nerves were well-myelinated 2 months after surgery. Regenerating nerves were on average 2.45 and 2.75 microm in diameter at 2 and 4 months, respectively, after surgery. These results indicate that the chitosan gel sponge sandwich might be suitable as a graft for peripheral nerve regeneration.     

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.     

Healing of periodontal defects treated with enamel matrix proteins and root surface conditioning--an experimental study in dogs

Application of enamel matrix proteins has been introduced as an alternative method for periodontal regenerative therapy. It is claimed that this approach provides periodontal regeneration by a biological approach, i.e. creating a matrix on the root surfaces that promotes cementum, periodontal ligament (PDL) and alveolar bone regeneration, thus mimicking the events occurring during tooth development. Although there have been numerous in vitro and in vivo studies demonstrating periodontal regeneration, acellular cementum formation and clinical outcomes via enamel matrix proteins usage, their effects on the healing pattern of soft and hard periodontal tissues are not well-established and compared with root conditioning alone. In the present study, the effects of Emdogain (Biora, Malm?, Sweden), an enamel matrix derivative mainly composed of enamel matrix proteins (test), on periodontal wound healing were evaluated and compared with root surface conditioning (performed with 36% orthophosphoric acid) alone (control) histopathologically and histomorphometrically by means of the soft and hard tissue profile of periodontium. An experimental periodontitis model performed at premolar teeth of four dogs were used in the study and the healing pattern of periodontal tissues was evaluated at days 7, 14, 21, 28 (one dog at each day), respectively. At day 7, soft tissue attachment evaluated by means of connective tissue and/or epithelial attachment to the root surfaces revealed higher connective tissue attachment rate in the test group and the amount of new connective tissue proliferation in the test group was significantly greater than the control group (p<0.01). New bone formation by osteoconduction initiated at day 14 in the test and control group. At day 21, the orientation of supra-alveolar and PDL fibers established, and new cementum formation observed in both groups. At day 28, although regenerated cementum was cellular in all of the roots in the control samples, an acellular type of cementum (1.32+/-0.83 mm in length and 3.16+/-0.23 microm in width) was also noted in six roots of test samples with an inconsistent distribution on the root surfaces. The amount of new cementum was significantly higher in the test group than the control group samples (p<0.01). The width of the cellular cementum in the control group was more than the cellular cementum in the test group, but the difference was not statistically significant (p>0.05). A firm attachment of acellular cementum to the root dentin with functional organization of its collagen fibers was noted, and, the accumulation and organization of cellular cementum in the control group was more irregular than the cellular cementum formed in the test group. The amount of new bone was 2.41+/-0.75 mm in the test and 1.09+/-0.46 mm in the control group at day 28. The rate of bone maturation (the number of osteons) was found higher in the test group (10.75+/-0.85) than the control group (5.50+/-0.86). Under the limitations of the study, our results reveal that when compared with root surface conditioning, enamel matrix proteins have more capacity for stimulating periodontal regeneration via their positive effects on root surfaces, i.e. inhibition of gingival epithelium down growth and stimulation of connective tissue proliferation and attachment to the root surfaces during wound healing. An acellular type of cementum regeneration and new alveolar bone formation by an accelerated osteoconductive mechanism are also achieved with application of enamel matrix proteins.     

Biomaterials in periodontal regenerative surgery: effects of cryopreserved bone, commercially available coral, demineralized freeze-dried dentin, and cementum on periodontal ligament fibroblasts and osteoblasts

The ultimate goal of periodontal therapy is to achieve successful periodontal regeneration. The effects of different biomaterials, allogenic and alloplastic, used in periodontal surgeries to achieve regeneration have been studied in vitro on periodontal ligament (PDL) cells and MC3T3-E1 cells. The materials tested included cryopreserved bone allograft (CBA), coralline hydroxyapatite (CH), demineralized freeze-dried dentin (DFDD), and cementum. CBA and CH revealed an increase in initial PDL cell attachment, whereas CH resulted in an increase in long-term PDL cell attachment. Mineral-like nodule formation was observed significantly higher in DFDD compared to other materials tested for osteoblasts. Based on the results of this in vitro study, we conclude that the materials used are all biocompatible with human PDL cells and osteoblasts, which have pivotal importance in periodontal regeneration.     

Culture of hepatocytes on fructose-modified chitosan scaffolds

Fructose was conjugated onto the inner surface of highly porous chitosan scaffold prepared by lyophilization. The modified scaffold with average pore size 50-200 microm was used to cultivate rat hepatocytes harvested by portal vein collagenase perfusion. The results indicated that while chitosan sponge alone supported cell attachment and growth, the scaffold modified with fructose accommodated a much larger number of hepatocytes due to the specific interaction between seeded hepatocytes and fructose moieties conjugated onto the surface of the scaffold. Hepatocytes exhibited a round cellular morphology with many microvilli evident on the surface of the cells, indicating healthy cells. Metabolic activities in terms of albumin secretion and urea synthesis were evaluated. It was found that hepatocytes cultured within fructose-modified scaffold resulted in much higher activities than within unmodified chitosan sponge. Scanning electron microscopy results showed that fructose-modified porous scaffold promoted the formation of cellular aggregates.     

Collagen Intermingled Chitosan-Tripolyphosphate Nano/Micro Fibrous Scaffolds for Tissue-Engineering Application

Abstract

Scaffolds comprising a nano- and micro-fibrous architecture are promising for tissue engineering, where nanofibers act as connecting network among microfibers and provide a 3D structural environment and mechanical stability for facilitating cell attachment, proliferation and migration. In this study, a novel structure was developed with polymeric micro and nano combined fibrous architecture, which aims to mimic the native extracellular matrix for tissue regeneration. Chitosan-tripolyphosphate (TPP) microfibers were prepared by wet spinning method, where collagen solution was allowed to self-assemble into nano/microfibers and subsequently freeze-dried for obtaining this combined architecture. To ensure prolonged mechanical stability, the scaffold was cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). This polymeric nano/micro combined scaffold revealed remarkable cellular activity and cytocompatibility towards both fibroblasts and osteoblast like cells and supported improved attachment and proliferation of cells than that of bare chitosan-TPP scaffolds owing to the presence of a bioactive molecule, collagen, in the intermingled form with chitosan-TPP microfiber.     

Bone regeneration on a collagen sponge self-assembled peptide-amphiphile nanofiber hybrid scaffold

The objective of this study was to create a novel approach to promote bone induction through sustained release of growth factor from a 3-dimensional (3D) hybrid scaffold. Peptide-amphiphile (PA) was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. Collagen sponge was reinforced by incorporation of poly(glycolic acid) (PGA) fiber. A 3D network of nanofibers was formed by mixing basic fibroblast growth factor (bFGF) suspensions with dilute aqueous solutions of PA. A hybrid scaffold was fabricated by combination of self-assembled PA nanofibers and collagen sponge reinforced with incorporation of PGA fibers. The in vitro release profile of bFGF from hybrid scaffold was investigated, and ectopic bone formation induced by the released bFGF was assessed after subcutaneous implantation of hybrid scaffold into the backs of rats. Homogeneous bone formation was histologically observed throughout the hybrid scaffolds, in marked contrast to collagen sponge-incorporated bFGF. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high compared with collagen sponge incorporated with bFGF. The combination of bFGF incorporated in a collagen sponge self-assembled PA nanofiber hybrid scaffold is a promising procedure to improve bone regeneration.     

Dual growth factor releasing multi-functional nanofibers for wound healing

The objective of this research is to develop a dual growth factor-releasing nanoparticle-in-nanofiber system for wound healing applications. In order to mimic and promote the natural healing procedure, chitosan and poly(ethylene oxide) were electrospun into nanofibrous meshes as mimics of extracellular matrix. Vascular endothelial growth factor (VEGF) was loaded within nanofibers to promote angiogenesis in the short term. In addition, platelet-derived growth factor-BB (PDGF-BB) encapsulated poly(lactic-co-glycolic acid) nanoparticles were embedded inside nanofibers to generate a sustained release of PDGF-BB for accelerated tissue regeneration and remodeling. In vitro studies revealed that our nanofibrous composites delivered VEGF quickly and PDGF-BB in a relayed manner, supported fibroblast growth and exhibited anti-bacterial activities. A preliminary in vivo study performed on normal full thickness rat skin wound models demonstrated that nanofiber/nanoparticle scaffolds significantly accelerated the wound healing process by promoting angiogenesis, increasing re-epithelialization and controlling granulation tissue formation. For later stages of healing, evidence also showed quicker collagen deposition and earlier remodeling of the injured site to achieve a faster full regeneration of skin compared to the commercial Hydrofera Blue® wound dressing. These results suggest that our nanoparticle-in-nanofiber system could provide a promising treatment for normal and chronic wound healing.     

Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds

Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering.     

Effects of preparation methods on the bone formation potential of apatite-coated chitosan microspheres

To investigate the effects of preparation methods on the bone formation potential of apatite-coated chitosan microspheres, coacervate precipitation method and emulsion cross-linking method were chosen to prepare chitosan microspheres, and then apatite coatings were deposited using simulated body fluid. Rat bone marrow-derived mesenchymal stem cells (BMSCs) were seeded on these microspheres. Cell adhesion, proliferation, and differentiation potential were monitored. For in vivo analysis, some cell/microsphere constructs were implanted in the subcutaneous pockets of male Wistar rats. After 3, 6, 12 weeks, the samples were retrieved and stained with hematoxylin and eosin (HE). Some cell/microsphere constructs were implanted in the calvarial defects of rats. Micro-CT and HE analysis were performed to analyze the new bone formation. It was found that BMSCs on apatite-coated emulsion cross-linked microspheres (EM1) exhibited better proliferation and differentiation than cells on apatite-coated coacervate-precipitated microspheres. The in vivo results showed that no bone was observed in ectopic areas. While in calvarial defects, both histological slices and Micro-CT images demonstrated that a substantial amount of new bone was formed in the EM1/BMSCs construct. These data suggest that preparation methods do exert great influence on the in vitro cell behaviors and in vivo orthotopic bone regeneration of apatite-coated chitosan microspheres. Appropriate method should be considered when preparing chitosan microspheres for bone tissue engineering scaffold.     

The effect of a bioactive collagen membrane releasing PDGF or GDF-5 on bone regeneration

Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using μCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative μCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.