Taking tissue-engineering principles into theater: augmentation of impacted allograft with human bone marrow stromal cells

Human bone marrow contains bone progenitor cells that arise from multipotent mesenchymal stem cells. Seeding bone progenitor cells onto a scaffold can produce a 3D living composite with significant mechanical and biological potential. This article details laboratory and clinical findings from two clinical cases, where different proximal femoral conditions were treated using impacted allograft augmented with marrow-derived autogenous progenitor cells. Autologous bone marrow was seeded onto highly washed morselized allograft and impacted. Samples of the impacted graft were also taken for ex vivo analysis. Both patients made an uncomplicated clinical recovery. Imaging confirmed defect filling with encouraging initial graft incorporation. Histochemical and alkaline phosphatase staining demonstrated that a live composite graft with osteogenic activity had been introduced into the defects. These studies demonstrate that marrow-derived cells can adhere to highly washed morselized allograft, survive the impaction process and proliferate with an osteoblastic phenotype, thus creating a living composite.     

Differential effect of a bone morphogenetic protein-7 (OP-1) on primary and revision loaded, stable implants with allograft

Morselized impacted bone allograft is often used to reconstruct the bone bed in the revision of failed total joint arthroplasties. We hypothesized that addition of the bone morphogenetic protein OP-1 (BMP-7) to bone allograft would improve early implant fixation. We inserted one loaded 6-mm-diameter titanium implant (surrounded by 0.75-mm gap) in each medial condyle of 24 canines. On one side, the implant was inserted in a controlled experimental revision setting resembling the clinical revision situation. A primary implant was inserted on the contralateral side in a previously unoperated site. Three groups were studied: 1) allograft alone, 2) allograft + 0.4 mg OP-1, and 3) allograft + 0.8 mg OP-1. Implant fixation was evaluated at 4 weeks. Grafted implants inserted in the primary setting without OP-1 had better fixation than the grafted revision setting with or without OP-1 (significantly more bone coverage, more mineralized tissue in the gap, and better mechanical interface strength). However, grafted primary implants with OP-1 had impaired fixation compared with grafted primary implants without OP-1 (less bone coverage of the implant and less bone formation in the gap). In contrast, grafted revision implants with OP-1 significantly increased implant fixation compared with grafted revision implants without OP-1 (increased mechanical interface strength and fraction of mineralized tissue in the gap). We found no differences between the two doses in any of the settings. Addition of OP-1 to bone allografted implants may show benefit at sites with impaired bone healing capacities, such as the revision setting.     

Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material,consisting of poly-l-lactide,carbon nanotubes and microhydroxyapatite,in the presence of bone morphogenetic protein-2

In bone tissue engineering composite materials have been introduced, combining a degradable polymer matrix with, for instance, carbon nanotubes (CNTs) to improve mechanical properties or with microhydroxyapatite (μHA) to improve osteoconduction. The addition of bone morphogenetic protein-2 (BMP-2) can further improve the biological response to the material. However, the influence of such an elaborate composite formation on osteoprogenitor cells is unknown.To examine this, rat bone marrow (RBM) cells were cultured on porous poly-l-lactic acid and composite scaffolds, with or without added BMP-2. Cell proliferation and differentiation were studied using DNA, alkaline phosphatase and scanning electron microscopic analysis. Further, genetic profiles were examined by microarray investigation. Results showed that the composite scaffold had no significant effect on the proliferation of RBM cells, but indicated a negative effect on cell differentiation. The addition of BMP-2 also had no significant effect on the proliferation of RBM cells, but differentiation towards the osteogenic lineage was confirmed. In the arrays results, the addition of BMP-2 alone led to the expression of genes involved in (minor) inflammation. The composite scaffold, and even more distinctly the combination of the composite scaffold with BMP-2, led to the expression of genes, based on gene ontology, connected to tumorigenesis. Therefore, CNT- and μHA-containing composite materials are not recommended as a bone restorative material.     

Bottom-up approach to construct microfabricated multi-layer scaffolds for bone tissue engineering

The use of bottom-up approaches in tissue engineering applications is advantageous since they enable the combination of various layers that could be made from different materials and/or incorporate different biochemical cues. Regarding the complex structure and the vascular system of the bone tissue, the aim of this study was to develop an innovative bottom-up approach that allows the construction of 3D biodegradable scaffolds from 2D microfabricated membranes with precise shape, pore size and porosity. For that purpose, poly (caprolactone) (PCL) and starch – poly (caprolactone) (SPCL (30 % starch)) blended sheets were used as substrates to produce the microfabricated membranes using micro hot-embossing. The use of this micro fabrication process allowed accurately imprinting micropillars and microholes in reproducible way. The assembling of the microfabricated membranes was performed using an easy, highly reproducible and inexpensive approach based on its successive stacking. Additionaly, the suitability of the microfabricated membranes to support the attachment and the cytoskeletal organization of human bone marrow stem cells (hBMSCs), macrovascular endothelial cells and osteoblasts derived from hBMSCs was demonstrated. Furthermore, hBMSCs proliferated and maintained the expression of the stromal progenitor marker STRO-1 when cultured on both PCL and SPCL microfabricated membranes. The proposed methodology constitutes a promising alternative to the traditional processing methods used to prepare tissue engineering scaffolds.     

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.     

骨组织工程支架材料的研究现状与应用前景

背景：目前组织工程骨修复骨缺损在临床应用中较为关键的问题是建立血管网,为新骨的形成提供氧气及营养物质,并为机体提供代谢途径。 目的：综述近年组织工程骨支架材料的特点,并着重介绍复合支架材料的研究现状。 方法：以“骨组织工程,血管化,支架材料,复合支架材料”为中文检索词,以“bone tissue engineering, vascularization,scaffold,composite scaffold”英文检索词,应用计算机在中国期刊全文数据库和PubMed数据库检索2001年1月至2014年1月的相关文章,将所有文章进行初步筛选后,对保留的文章进一步详细分析、归纳并总结。 结果与结论：按照组织工程骨支架材料的来源不同,可将其分为人工合成材料、天然衍生材料和复合支架材料,单一支架材料难以作为最理想的材料修复骨缺损,复合支架材料能在不同程度上弥补单一支架材料的缺陷,因此近年来组织工程支架材料的发展由单一材料发展为复合材料,并呈现人工合成材料与天然材料有机结合的趋势。但复合支架材料在临床应用中仍然有许多尚待解决的问题,主要有控制复合材料比例,使材料降解速率与组织细胞的生长速率相适应,保持复合材料的多孔隙和高机械强度。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering

Biodegradable polymers and bioactive ceramics are being combined in a variety of composite materials for tissue engineering scaffolds. Materials and fabrication routes for three-dimensional (3D) scaffolds with interconnected high porosities suitable for bone tissue engineering are reviewed. Different polymer and ceramic compositions applied and their impact on biodegradability and bioactivity of the scaffolds are discussed, including in vitro and in vivo assessments. The mechanical properties of today's available porous scaffolds are analyzed in detail, revealing insufficient elastic stiffness and compressive strength compared to human bone. Further challenges in scaffold fabrication for tissue engineering such as biomolecules incorporation, surface functionalization and 3D scaffold characterization are discussed, giving possible solution strategies. Stem cell incorporation into scaffolds as a future trend is addressed shortly, highlighting the immense potential for creating next-generation synthetic/living composite biomaterials that feature high adaptiveness to the biological environment.     

Three-dimensional,bioactive, biodegradable,polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro

In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biological and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft. Because no single existing material possesses all the necessary properties required in an ideal bone graft, our approach has been to develop a three dimensional (3-D), porous composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) that is biodegradable, bioactive, and suitable as a scaffold for bone tissue engineering (PLAGA-BG composite). The objectives of this study were to examine the mechanical properties of a PLAGA-BG matrix, to evaluate the response of human osteoblast-like cells to the PLAGA-BG composite, and to evaluate the ability of the composite to form a surface calcium phosphate layer in vitro. Structural and mechanical properties of PLAGA-BG were measured, and the formation of a surface calcium phosphate layer was evaluated by surface analysis methods. The growth and differentiation of human osteoblast-like cells on PLAGA-BG were also examined. A hypothesis was that the combination of PLAGA with BG would result in a biocompatible and bioactive composite, capable of supporting osteoblast adhesion, growth and differentiation, with mechanical properties superior to PLAGA alone. The addition of bioactive glass granules to the PLAGA matrix resulted in a structure with higher compressive modulus than PLAGA alone. Moreover, the PLAGA-BA composite was found to be a bioactive material, as it formed surface calcium phosphate deposits in a simulated body fluid (SBF), and in the presence of cells and serum proteins. The composite supported osteoblast-like morphology, stained positively for alkaline phosphatase, and supported higher levels of Type I collagen synthesis than tissue culture polystyrene controls. We have successfully developed a degradable, porous, polymer bioactive glass composite possessing improved mechanical properties and osteointegrative potential compared to degradable polymers of poly(lactic acid-glycolic acid) alone. Future work will focus on the optimization of the composite scaffold for bone tissue-engineering applications and the evaluation of the 3-D composite in an in vivo model.     

The application of human bone marrow stromal cells and poly(dl-lactic acid) as a biological bone graft extender in impaction bone grafting

Concerns over disease transmission, high costs and limited supply have led to interest in synthetic grafts in the field of impaction bone grafting (IBG). Poly(DL-lactic acid) (PLA) grafts are attractive alternatives due to their biocompatibility, established safety and versatile manufacturing process. This study examined the potential of PLA scaffolds augmented with human bone marrow stromal cells (HBMSCs) in IBG. In vitro and in vivo studies were performed on impacted morsellised PLA seeded with HBMSC and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, for 4 weeks. Biochemical, histological, mechanical and 3D micro-computed tomography analyses were performed. HBMSC viability, biochemical activity and histological evidence of osteogenic cellular differentiation, post-impaction were observed in vitro and in vivo in PLA/HBMSC samples compared to impacted PLA alone. In vitro PLA/HBMSC samples demonstrated evidence of mechanical enhancement over PLA alone. In vivo studies showed a significant increase in new bone and blood vessel formation in the PLA/HBMSC constructs compared to PLA alone. With alternatives to allograft being sought, these studies have demonstrated PLA/HBMSC living composites, to be a potential prospect as a biological bone graft extender for future use in the field of IBG.     

Effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells in PHBV/wollastonite composite scaffolds

In this study, the effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells (hBMSCs) have been investigated based on a polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/ wollastonite (W) composite scaffolds system. Cell morphology, proliferation, and differentiation were measured. The results showed that the incorporation of wollastonite benefited hBMSCs adhesion, proliferation, and differentiation rate. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composite scaffolds increased from 10 to 20 wt%. Based on our previous studies on PHBV/W composite discs, the differentiation measurements in this paper further proved that the wollastonite itself can stimulate the hBMSCs to differentiate toward osteoblasts without any osteogenic medium, and the ionic products (Ca and Si) released from wollastonite might contribute to this advantage. It is also suggested that the osteogenic differentiation of the hBMSCs can be affected by adjusting the wollastonite content in the composite scaffolds.     

Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects

Reconstruction of large area bone defect with mechanical integrity to the skeleton is important for patient's rehabilitation. However with the limitation of scaffold material and suitable seed cell sources, the best treating strategy remains to be identified though various tissue engineering methods were reported. In this study, we investigated the feasibility of applying calcined bovine bone (CBB) which was coated by allograft bone marrow mesenchymal stem cells (BMSC)-sheet as a 3D scaffold material in bone repairing tissue engineering. The new scaffold material was implanted into osteoporosis rat cranial bone defects and repairing critical size bone defects (8 mm diameter). Data showed that CBB-BMSC-sheet combination had a stronger potential in osteogenic differentiation and mineralized formation both in vitro and in vivo than CBB-BMSC combination. In in vitro study BMSC-sheet had a more feasible characteristic upon bone repairing including richer ECM, larger mineralized area and stronger ALP activity in addition with a significant higher mRNA expression of osteogenic maker such as BMP-2, b-FGF, Col 1a1, OSX and Runx-2 than the control group. In in vivo study 3D reconstruction of micro CT, HE staining and bone strength results showed that newly formed bone in CBB-BMSC-sheet group was significant higher than that in CBB-BMSC group at 4, 8 and 12 weeks after transplantation in the aspect of area and volume. What was more, results indicated that allograft BMSC-sheet had survivaled in the scaffold material and participated in the newly formed bone which had the same thickness with surrounding autologous bone tissues after transplantation. Results of our study demonstrated that CBB-BMSC-sheet combination was a promising strategy in healing of large area bone defect in osteoporosis.     

Superior mineralization and neovascularization capacity of adult human metaphyseal periosteum-derived cells for skeletal tissue engineering applications

Bone tissue engineering is a promising cell-based strategy to treat bone defects. Mesenchymal stem cells from adult human bone marrow (hBMSCs) are a frequently used cellular source for bone tissue generation. However, the low frequency of these stem cells in adult bone marrow and their limited proliferation restrict their clinical utility. An alternative source of MSCs is the periosteum-derived cells, and these cells appear to be easy to harvest and expand ex vivo. We isolated human metaphyseal periosteum-derived cells (hMPCs) and hBMSCs from the same donors and compared their osteogenic capacity both in vitro and in vivo. After osteogenic induction in monolayer cultures, hMPCs resulted in more robust mineralization and expressed higher mRNA levels of BMP-2, osteopontin and osteocalcin than hBMSCs. Eight weeks after implantation of cellular-β-TCP scaffolds in immunodeficient mice, hMPC implantation showed higher neovascularization and higher percentage of mature bone formation than hBMSC implantation. In conclusion, hMPCs represent a promising cellular candidate for bone tissue engineering.     

Poly-epsilon-caprolactone/hydroxyapatite composites for bone regeneration: in vitro characterization and human osteoblast response

Polycaprolactone (PCL), a semicrystalline linear resorbable aliphatic polyester, is a good candidate as a scaffold for bone tissue engineering, due to its biocompatibility and biodegradability. However, the poor mechanical properties of PCL impair its use as scaffold for hard tissue regeneration, unless mechanical reinforcement is provided. To enhance mechanical properties and promote osteoconductivity, hydroxyapatite (HA) particles were added to the PCL matrix: three PCL-based composites with different volume ratio of HA (13%, 20%, and 32%) were studied. Mechanical properties and structure were analysed, along with biocompatibility and osteoconductivity. The addition of HA particles (in particular in the range of 20% and 32%) led to a significant improvement in mechanical performance (e.g., elastic modulus) of scaffold. Saos-2 cells and osteoblasts from human trabecular bone (hOB) retrieved during total hip replacement surgery were seeded onto 3D PCL samples for 1-4 weeks. Following the assessment of cell viability, proliferation, morphology, and ALP release, HA-loaded PCL was found to improve osteoconduction compared to the PCL alone. The results indicated that PCL represents a potential candidate as an efficient substrate for bone substitution through an accurate balance between structural/ mechanical properties of polymer and biological activities.     

可注射型组织工程骨支架材料的研究进展

可注射型组织工程骨支架材料是一种具有一定形态和机械强度的支架材料,可与种子细胞复合,以流体的形式注射到骨组织缺损部位,最终形成新骨,达到结构恢复和功能重建的目的.此材料具有创伤小、可塑性好的特点,可以修复形态不规则的骨缺损,能够很好地复合生长因子,是目前较为理想的骨组织缺损的修复方式.在众多可注射骨组织工程材料中,生物陶瓷材料、高分子材料等被证明有高度的生物相容性和良好的机械性能,已成为骨组织工程材料方面的研究重点.旨在对生物陶瓷材料、高分子材料、生物陶瓷与高分子复合材料的发展与应用作一综述.     

Morsellised sawbones is an acceptable experimental substitute for the in vitro elastic and viscoelastic mechanical characterisation of morsellised cancellous bone undergoing impaction grafting

Impaction grafting using morsellised bone chips is widely used during surgery to mitigate the effects of bone loss. The technique typically involves the packing of morsellised allograft cancellous bone into bone defects, and has found extensive application in revision hip and knee surgery. In the ideal situation, the presence of the bone graft prevents subsidence of the revised prosthesis in the short term, and integrates with the host bone in the longer term. However, the configuration of particles within the graft remains to be optimised, and is highly likely to vary across potential sites and loading conditions. Human bone, for use in experimental investigation, is often difficult to obtain with properties that are relevant from a clinical point of view. This study, therefore, has explored the mechanical response of a Sawbones based experimental substitute. An established confined compression technique was used to characterise the morsellised Sawbones material. Comparison of the results with published values for bovine and human bone indicate that the mechanical response of the morsellised Sawbones material map well onto the elastic and viscoelastic response of bone of a biological origin.     

Repair of a proximal femoral bone defect in dogs using a porous surfaced prosthesis in combination with recombinant BMP-2 and a synthetic polymer carrier

Total hip arthroplasty (THA) has become an almost standard procedure for the treatment of various hip lesions. However, one of the limitations has been the mechanical loosening of the prosthesis, a condition termed peri-prosthetic osteolysis. Consequently, at revision surgery, various grades of bone defect are often noted. Alternative approaches aimed at overcoming this problem have included a special design of the revision prosthesis and allo- or autogeneic bone grafting in combination with or without biomaterials. In a further attempt to address the loosening of the prosthesis, we have combined human bone morphogenetic protein-2, produced by DNA recombination (rhBMP-2) with a new synthetic biodegradable polymer (poly-D,L-lactic-acid-para-dioxanone-polyethyleneglycol block co-polymer; PLA-DX-PEG). We present data on the efficacy of the rhBMP-2 laden prosthesis to reconstruct a bone defect in a canine model. In this model, medial half of the proximal femur was surgically resected to create a bone defect that was repaired with the rhBMP-2/PLA-DX-PEG composite. Twelve weeks after implantation, the original bone defects in the rhBMP-2 treatment groups had been repaired. Thus, this type of 'hybrid' prosthesis may provide a new modality to repair bone defects or restore lost bone mass encountered in revision arthroplasty.     

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.     

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.     

Influence of a novel calcium-phosphate coating on the mechanical properties of highly porous collagen scaffolds for bone repair

Lyophilised collagen scaffolds have shown enormous potential in tissue engineering in a number of areas due to their excellent biological performance. However, they are limited for use in bone tissue engineering due to poor mechanical properties. This paper discusses the development of a calcium-phosphate coating for collagen scaffolds in order to improve their mechanical properties for bone tissue engineering.Pure collagen scaffolds produced in a lyophilisation process were coated by immersing them in sodium ammonium hydrogen phosphate (NaNH₄HPO₄) followed by calcium chloride (CaCl₂). The optimal immersing sequence, duration, as well as the optimal solution concentration which facilitated improved mechanical properties of the scaffolds was investigated. The influence of the coating on composition, structural and material properties was analysed.This investigation successfully developed a novel collagen/calcium-phosphate composite scaffold. An increase in the mechanical properties of the scaffolds from 0.3 kPa to up to 90 kPa was found relative to a pure collagen scaffold, while the porosity was maintained as high as 92%, indicating the potential of the scaffold for bone tissue engineering or as a bone graft substitute.