Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ～ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.     

Three-dimensional scaffold containing EGF incorporated biodegradable polymeric nanoparticles for stem cell based tissue engineering applications

Stem cell-based tissue engineering holds much hope for the development of multifunctional tissues to replace diseased organs. The attachment and survival of stem cells on a three-dimensional (3D) scaffold must be enhanced for faster progression of stem cell based tissue engineering. This study evaluate the stability of mesenchymal stem cells (MSCs) in 3D porous scaffolds composed of a collagen and chitosan blend impregnated with epidermal growth factor incorporated chitosan nanoparticles (EGF-CNP). The EGF-CNP scaffolds were characterized by transmission electron microscopy, which revealed that the nanoparticles were round in shape and 20 ∼ 50 nm in size. The scaffolds were prepared by freeze drying. A Fourier-transform infrared spectrum study revealed that the linkage between collagen and chitosan was through an ionic interaction. Thermal analysis and degradation studies showed that the scaffold could be used in tissue engineering application. MSCs proliferated well in the EGF-CNP impregnated scaffold. A scanning electron microscope study showed anchored and elongated MSCs on the EGF-CNP impregnated scaffold. A 3D biodegradable collagen chitosan scaffold impregnated with EGF-CNP is a promising transportable candidate for MSC-based tissue engineering, and this scaffold could be used as an in vitro model for subsequent clinical applications.     

生物支架材料——组织工程连载之二

具有三维结构的支架材料是组织工程的核心内容之一。现有组织工程支架可分为天然生物材料、合成有机材料和无机材料三类。支架材料近年来研究十分活跃,不仅在组织工程的最早产品人工皮肤领域进行了更为完善的研究和开发,同时在诸如人工骨、软骨、神经、血管、皮肤、肝、脾、肾、膀胱等方面进行了大量研究和探索。与普通组织工程支架需要预先制备并在体外成型不同,近年来在骨和软骨组织工程实践中兴起的可注射支架具有许多优势,是未来组织工程支架发展的重要方向之一。     

Repair and regeneration of osteochondral defects in the articular joints

People suffering from pain due to osteoarthritic or rheumatoidal changes in the joints are still waiting for a better treatment. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of osteochondral defects. Also joint replacements have not yet succeeded in replacing of natural cartilage without complications. Therefore, there is room for a new medical approach, which outperforms currently used methods. The aim of this study is to show potential of using a tissue engineering approach for regeneration of osteochondral defects. The critical review of currently used methods for treatment of osteochondral defects is also provided. In this study, two kinds of hybrid scaffolds developed in Hutmacher's group have been analysed. The first biphasic scaffold consists of fibrin and PCL. The fibrin serves as a cartilage phase while the porous PCL scaffold acts as the subchondral phase. The second system comprises of PCL and PCL-TCP. The scaffolds were fabricated via fused deposition modeling which is a rapid prototyping system. Bone marrow-derived mesenchymal cells were isolated from New Zealand White rabbits, cultured in vitro and seeded into the scaffolds. Bone regenerations of the subchondral phases were quantified via micro CT analysis and the results demonstrated the potential of the porous PCL and PCL-TCP scaffolds in promoting bone healing. Fibrin was found to be lacking in this aspect as it degrades rapidly. On the other hand, the porous PCL scaffold degrades slowly hence it provides an effective mechanical support. This study shows that in the field of cartilage repair or replacement, tissue engineering may have big impact in the future. In vivo bone and cartilage engineering via combining a novel composite, biphasic scaffold technology with a MSC has been shown a high potential in the knee defect regeneration in the animal models. However, the clinical application of tissue engineering requires the future research work due to several problems, such as scaffold design, cellular delivery and implantation strategies.     

骨组织工程天然衍生细胞外基质材料

细胞外基质材料的开发是骨组织工程的重要组成部分,目前,在骨组织工程中应用较多的基质材料可分为天然衍生材料、人工合成材料以及这两种材料的复合材料。介绍了各种天然衍生骨材料如煅烧骨、脱钙骨基质、脱蛋白骨基质、重组合异种骨基质和天然高分子材料如胶原、纤维蛋白、几丁质、藻酸盐及其衍生物以及珊瑚衍生骨在骨组织工程中的应用,展望了骨组织工程细胞外基质材料的未来发展方向,认为未来的理想基质材料应该是集各种材料的优点于一身,能够充分适应体内各种生理环境并能采用智能化的加工方式进行大批量生产的生物仿生材料。     

Polyvinyl alcohol modified polyvinylidene fluoride-graphene oxide scaffold promotes osteogenic differentiation potential of human induced pluripotent stem cells

Tissue engineering is fast becoming a key approach in bone medicine studies. Designing the ideally desirable combination of stem cells and scaffolds are at the hurt of efforts for producing implantable bone substitutes. Clinical application of stem cells could be associated with serious limitations, and engineering scaffolds that are able to imitate the important features of extracellular matrix is a major area of challenges within the field. In this study, electrospun scaffolds of polyvinylidene fluoride (PVDF), PVDF-graphene oxide (GO), PVDF-polyvinyl alcohol (PVA) and PVDF-PVA-GO were fabricated to study the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs) while cultured on fabricated scaffolds. Scanning electron microscopy study, viability assay, relative gene expression analysis, immunocytochemistry, alkaline phosphates activity, and calcium content assays confirmed that the osteogenesis rate of hiPSCs cultured on PVDF-PVA-Go is significantly higher than other scaffolds. Here, we showed that the biocompatible, nontoxic, flexible, piezoelectric, highly porous and interconnected three-dimensional structure of electrospun PVDF-PVA-Go scaffold in combination with hiPSCs (as the stem cells with significant advantageous in comparison to other types) makes them a highly promising scaffold-stem cell system for bone remodeling medicine. There was no evidence for the superiority of PVDF-GO or PVDF-PVA scaffold for osteogenesis, compared to each other; however both of them showed better potentials as to PVDF scaffold.     

Finite element method (FEM), mechanobiology and biomimetic scaffolds in bone tissue engineering

Techniques of bone reconstructive surgery are largely based on conventional, non-cell-based therapies that rely on the use of durable materials from outside the patient's body. In contrast to conventional materials, bone tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve bone tissue function. Bone tissue engineering has led to great expectations for clinical surgery or various diseases that cannot be solved with traditional devices. For example, critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of bone tissue engineering is to apply engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. The total market for bone tissue regeneration and repair was valued at $1.1 billion in 2007 and is projected to increase to nearly $1.6 billion by 2014.Usually, temporary biomimetic scaffolds are utilized for accommodating cell growth and bone tissue genesis. The scaffold has to promote biological processes such as the production of extra-cellular matrix and vascularisation, furthermore the scaffold has to withstand the mechanical loads acting on it and to transfer them to the natural tissues located in the vicinity. The design of a scaffold for the guided regeneration of a bony tissue requires a multidisciplinary approach. Finite element method and mechanobiology can be used in an integrated approach to find the optimal parameters governing bone scaffold performance.In this paper, a review of the studies that through a combined use of finite element method and mechano-regulation algorithms described the possible patterns of tissue differentiation in biomimetic scaffolds for bone tissue engineering is given. Firstly, the generalities of the finite element method of structural analysis are outlined; second, the issues related to the generation of a finite element model of a given anatomical site or of a bone scaffold are discussed; thirdly, the principles on which mechanobiology is based, the principal theories as well as the main applications of mechano-regulation models in bone tissue engineering are described; finally, the limitations of the mechanobiological models and the future perspectives are indicated.     

Computational modeling of flow-induced shear stresses within 3D salt-leached porous scaffolds imaged via micro-CT

Flow-induced shear stresses have been found to be a stimulatory factor in pre-osteoblastic cells seeded in 3D porous scaffolds and cultured under continuous flow perfusion. However, due to the complex internal structure of porous scaffolds, analytical estimation of the local shear forces is impractical. The primary goal of this work is to investigate the shear stress distributions within Poly(l-lactic acid) scaffolds via computation. Scaffolds used in this study are prepared via salt leeching with various geometric characteristics (80–95% porosity and 215–402.5 μm average pore size). High resolution micro-computed tomography is used to obtain their 3D structure. Flow of osteogenic media through the scaffolds is modeled via lattice Boltzmann method. It is found that the surface stress distributions within the scaffolds are characterized by long tails to the right (a positive skewness). Their shape is not strongly dependent on the scaffold manufacturing parameters, but the magnitudes of the stresses are. Correlations are prepared for the estimation of the average surface shear stress experienced by the cells within the scaffolds and of the probability density function of the surface stresses. Though the manufacturing technique does not appear to affect the shape of the shear stress distributions, presence of manufacturing defects is found to be significant: defects create areas of high flow and high stress along their periphery. The results of this study are applicable to other polymer systems provided that they are manufactured by a similar salt leeching technique, while the imaging/modeling approach is applicable to all scaffolds relevant to tissue engineering.     

Establishment of a three-dimensional culture and mechanical loading system for skeletal myoblasts

Establishment of a three-dimensional (3-D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four-point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60-88 μm), type II (88-100 μm) and type III (100-125 μm). To establish the 3-D mechanical loading system, PLGA membrane was integrated with conventional force-loading plates and the third passage skeletal myoblasts from neonatal Sprague-Dawley (SD) rats were seeded. Small PLGA membranes were put in 24-well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days’ culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 μstrain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3-D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four-point bending strain unit provides a satisfactory 3-D mechanical loading system.     

生物衍生松质骨支架材料的Micro—CT量化分析

目的：在骨组织工程中,如何制备出理想的支架材料一直是研究重点;目前主要的有天然生物支架材料、人工合成有机材料和无机材料等;生物衍生骨即天然生物支架材料的一种,由于其与天然骨在形态结构上较为相似,是近年来研究较多的支架材料之一;既往形态学研究局限于在二维层面,对于其三维结构参数分析较少。故本实验主要运用Micro-CT对生物衍生松质骨的三维结构参数进行分析,量化评价其作为骨组织工程支架材料的结构参数。方法：截取新鲜猪松质骨,经脱脂脱蛋白部分脱钙及去抗原处理后,制作成生物衍生骨支架;应用Micro．CT扫描,重建三维图并量化分析其结构参数,统计软件SPSS分析各参数间的相关性。结果：经Micro．CT扫描,得到二维CT图和三维重建图。各三维结构参数的值分别为：BV／TV（20．48±5．14）％;BS／BV（41．66±5．39）1／ram;Porosity（79．52±5．14）％;Tb．Th（0．10±0．01）mm;Tb．N（1．99±0．47）l／mm;Tb．Sp（0．32±0．05）mm;Tb．Pf（2．03±4．70）1／mm;SMI（1．28±0．35）;DA（1．60±0．23）;Corm．Dn（158．53±106．09）I／mm3。各参数间相关系数具有统计学意义的为：（1）Porosity与BS／BV、Tb．Th;（2）BV／TV与BS／BV、Tb．Th;（3）BS／BV与Coma．Dn、Porosity、BV/TV、;（4）Tb．Th与Porosity、BV厂IV、Conn．Dn;（5）DA与Corm．Dn;（6）Conn．Dn与Bs／BV、Tb．Th、DA。结论：Micro-CT扫描、量化分析是评价支架材料结构参数的理想方法;也证明生物衍生骨支架符合骨组织工程对支架材料的三维结构要求,尤其在孔径大小、孔隙率、表面积体积比等三维结构参数,此外,也可为其他支架材料的制备在三维结构上的要求提供参考依据。     

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

Naringin‐inlaid silk fibroin/hydroxyapatite scaffold enhances human umbilical cord‐derived mesenchymal stem cell‐based bone regeneration

ObjectivesLarge bone defects are a common, debilitating clinical condition that have substantial global health and economic burden. Bone tissue engineering technology has become one of the most promising approaches for regenerating defective bones. In this study, we fabricated a naringin‐inlaid composite silk fibroin/hydroxyapatite (NG/SF/HAp) scaffold to repair bone defects.Materials and MethodsThe salt‐leaching technology was used to fabricate the NG/SF/HAp scaffold. The cytocompatibility of the NG/SF/HAp scaffold was assessed using scanning electron microscopy, live/dead cell staining and phalloidin staining. The osteogenic and angiogenic properties were assessed in vitro and in vivo.ResultsThe porous NG/SF/HAp scaffold had a well‐designed biomimetic porous structure with osteoinductive and angiogenic activities. A gene microarray identified 854 differentially expressed genes between human umbilical cord‐derived mesenchymal stem cells (hUCMSCs) cultured on SF‐nHAp scaffolds and cells cultured on NG/SF/HAp scaffolds. The underlying osteoblastic mechanism was investigated using hUCMSCs in vitro. Naringin facilitated hUCMSC ingrowth into the SF/HAp scaffold and promoted osteogenic differentiation. The osteogenic and angiogenic capabilities of cells cultured in the NG/SF/HAp scaffold were superior to those of cells cultured in the SF/HAp scaffold.ConclusionsThe data indicate the potential of the SF/HAp composite scaffold incorporating naringin for bone regeneration.     

Review: Development of clinically relevant scaffolds for vascularised bone tissue engineering

Clinical translation of scaffold-based bone tissue engineering (BTE) therapy still faces many challenges despite intense investigations and advancement over the years. To address these clinical barriers, it is important to analyse the current technical challenges in constructing a clinically relevant scaffold and subsequent clinical issues relating to bone repair. This review highlights the key challenges hampering widespread clinical translation of scaffold-based vascularised BTE, with a focus on the repair of large non-union defects. The main limitations of current scaffolds include the lack of sufficient vascularisation, insufficient mechanical strength as well as issues relating to the osseointegration of the bioresorbable scaffold and bone infection management. Critical insights on the current trends of scaffold technologies and future directions for advancing next-generation BTE scaffolds into the clinical realm are discussed. Considerations concerning regulatory approval and the route towards commercialisation of the scaffolds for widespread clinical utility will also be introduced.     

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.     

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three‐dimensional scaffold from nano‐hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X‐ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM‐MSCs)/scaffold. After 1, 4, and 12 weeks post‐injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin–eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM‐MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre‐seeded nHA/Gel/SiC scaffold with rBM‐MSCs improves osteogenesis in the engineered bone implant.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

Magnetic hydroxyapatite bone substitutes to enhance tissue regeneration: evaluation in vitro using osteoblast-like cells and in vivo in a bone defect

In case of degenerative disease or lesion, bone tissue replacement and regeneration is an important clinical goal. In particular, nowadays, critical size defects rely on the engineering of scaffolds that are 3D structural supports, allowing cellular infiltration and subsequent integration with the native tissue. Several ceramic hydroxyapatite (HA) scaffolds with high porosity and good osteointegration have been developed in the past few decades but they have not solved completely the problems related to bone defects. In the present study we have developed a novel porous ceramic composite made of HA that incorporates magnetite at three different ratios: HA/Mgn 95/5, HA/Mgn 90/10 and HA/Mgn 50/50. The scaffolds, consolidated by sintering at high temperature in a controlled atmosphere, have been analysed in vitro using human osteoblast-like cells. Results indicate high biocompatibility, similar to a commercially available HA bone graft, with no negative effects arising from the presence of magnetite or by the use of a static magnetic field. HA/Mgn 90/10 was shown to enhance cell proliferation at the early stage. Moreover, it has been implanted in vivo in a critical size lesion of the rabbit condyle and a good level of histocompatibility was observed. Such results identify this scaffold as particularly relevant for bone tissue regeneration and open new perspectives for the application of a magnetic field in a clinical setting of bone replacement, either for magnetic scaffold fixation or magnetic drug delivery.     

Human Urine Derived Stem Cells in Combination with β-TCP Can Be Applied for Bone Regeneration

Bone tissue engineering requires highly proliferative stem cells that are easy to isolate. Human urine stem cells (USCs) are abundant and can be easily harvested without using an invasive procedure. In addition, in our previous studies, USCs have been proved to be able to differentiate into osteoblasts, chondrocytes, and adipocytes. Therefore, USCs may have great potential and advantages to be applied as a cell source for tissue engineering. However, there are no published studies that describe the interactions between USCs and biomaterials and applications of USCs for bone tissue engineering. Therefore, the objective of the present study was to evaluate the interactions between USCs with a typical bone tissue engineering scaffold, beta-Tricalcium Phosphate (β-TCP), and to determine whether the USCs seeded onto β-TCP scaffold can promote bone regeneration in a segmental femoral defect of rats. Primary USCs were isolated from urine and seeded on β-TCP scaffolds. Results showed that USCs remained viable and proliferated within β-TCP. The osteogenic differentiation of USCs within the scaffolds was demonstrated by increased alkaline phosphatase activity and calcium content. Furthermore, β-TCP with adherent USCs (USCs/β-TCP) were implanted in a 6-mm critical size femoral defect of rats for 12 weeks. Bone regeneration was determined using X-ray, micro-CT, and histologic analyses. Results further demonstrated that USCs in the scaffolds could enhance new bone formation, which spanned bone defects in 5 out of 11 rats while β-TCP scaffold alone induced modest bone formation. The current study indicated that the USCs can be used as a cell source for bone tissue engineering as they are compatible with bone tissue engineering scaffolds and can stimulate the regeneration of bone in a critical size bone defect.