Response of the donor and recipient cells in mesenchymal cell transplantation to cartilage defect

To facilitate the repair of articular cartilage defects, autologous mesenchymal cells from bone marrow or periosteum were transplanted in a rabbit model. Two weeks after the transplantation of the mesenchymal cells, the whole area of the original defect was occupied by cartilage. From the deep area of the reparative cartilage, which contacted with host bone, chondrocytes became hypertrophic and the invasion of bone with vasculature started, until the replacement reached the natural junction of the host cartilage and the subchondral bone about 4 weeks after transplantation. Twelve weeks after the transplantation, the repair cartilage in the defect became a little thinner than the adjacent normal cartilage, which became a little thinner 24 weeks after the transplantation (the longest observation period in the study). Large, full-thickness defects of the weight-bearing region of the articular cartilage were repaired with hyaline-like cartilage after implantation of autologous mesenchymal cells. The repair process by mesenchymal cell transplantation was explained as follows: The donor transplanted cell differentiated into cartilage and the defects were completely filled with cartilage. Then, mesenchymal cells that entered the chondrogenic lineage rapidly progressed through this lineage to the hypertrophic state, which was then the target for erosion and vascular invasion. Although this vasculature and the newly formed bone were considered to be host-derived, there was no evidence to that effect. To prove this, suitable experimental marking of these donor cells is needed. In the case of chondrocyte transplantation, the repair cartilage maintained its thickness to the full depth of the original defect; the tissue derived from the implanted chondrocytes was not invaded by vessels or replaced by subchondral bone.     

聚氨酯耳廓软骨支架的快速成形工艺

在体外构建外形逼真、机械强度可调节的个性化耳廓软骨支架,是组织工程方法修复外耳耳廓缺损的重要方向之一。针对这一研究目的,采用基于快速成形原理的熔融挤出成形方法,使用颗粒状聚氨酯生物材料,通过搭建试验平台,设计一系列工艺因素对成形过程及支架力学性能的影响试验,研究制造耳廓软骨支架的最优工艺参数范围。试验发现:喷头温度影响材料挤出稳定性和弹性;成形室温度影响结构整齐度和层间结合度;送料速度和扫描速度,与挤出速度的匹配关系,影响出丝的均匀度和成形过程的稳定性;不同扫描速度的喷头所形成的温度场分布,影响已成形结构的弹性;成形过程存在最小局部反复造型面积;温度以及CLI数字模型离散处理参数,影响支架力学性能。结果表明,熔融挤出成形方法制备的聚氨酯材料耳廓软骨支架,不仅可以实现逼真的耳廓外形,还可通过控制各种工艺参数,得到接近天然耳廓的力学强度,以满足临床中不同病患的个性化需求。     

Mechanical testing of recombinant human bone morphogenetic protein-7 regenerated bone in sheep mandibles

A new method was developed in this study for testing excised sheep mandibles as a cantilever. The method was used to determine the strength and stiffness of sheep hemi-mandibles including a 35 mm defect bridged by regenerated bone. Recombinant human bone morphogenetic protein-7 (rhBMP-7) in a bovine collagen type-I carrier was used for the bone regeneration. Initial tests on ten intact sheep mandibles confirmed that the strength, stiffness and area beneath the load-deformation curves of the right and left hemi-mandibles were not significantly different, confirming the validity of using the contra-lateral hemi-mandible as a control side. Complete bone regeneration occurred in six hemi-mandibles treated with rhBMP, but the quality and mechanical properties of the bone were very variable. The new bone in three samples contained fibrous tissue and was weaker and less stiff than the contra-lateral side (strength, 10-20 per cent; stiffness, 6-15 per cent). The other half had better-quality bone and was significantly stiffer and stronger (p < 0.05), with strength 45-63 per cent and stiffness 35-46 per cent of the contra-lateral side. Hemi-mandibles treated with collagen alone had no regenerated bone bridge suggesting that 35 mm is a critical-size bone defect.     

A finite element model of an idealized diarthrodial joint to investigate the effects of variation in the mechanical properties of the tissues

The stiffness of articular cartilage increases dramatically with increasing rate of loading, and it has been hypothesized that increasing the stiffness of the subchondral bone may result in damaging stresses being generated in the articular cartilage. Despite the interdependence of these tissues in a joint, little is understood of the effect of such changes in one tissue on stresses generated in another. To investigate this, a parametric finite element model of an idealized joint was developed. The model incorporated layers representing articular cartilage, calcified cartilage, the subchondral bone plate and cancellous bone. Taguchi factorial design techniques, employing a two-level full-factorial and a four-level fractional factorial design, were used to vary the material properties and thicknesses of the layers over the wide range of values found in the literature. The effects on the maximum values of von Mises stress in each of the tissues are reported here. The stiffness of the cartilage was the main factor that determined the stress in the articular cartilage. This, and the thickness of the cartilage, also had the largest effect on the stresses in all the other tissues with the exception of the subchondral bone plate, in which stresses were dominated by its own stiffness. The stiffness of the underlying subchondral bone had no effect on the stresses generated in the cartilage. This study shows how stresses in the various tissues are affected by changes in their mechanical properties and thicknesses. It also demonstrates the benefits of a structured, systematic approach to investigating parameter variation in finite element models.     

Behavior of graft and host cells in underlying subchondral bone after transplantation of osteochondral autograft

Transplantation of osteochondral autograft is widely used as a therapeutic strategy for the defect of articular cartilage. In the repair process, although underlying subchondral bone becomes necrotic and then is followed by bone reconstruction, the fate of graft and host cells during remodeling of underlying subchondral bone has not been elucidated. The objectives of this study were to establish a method to follow graft and host cells after transplantation of osteochondral autograft, and to elucidate the fate of both graft and host cells during remodeling of underlying subchondral bone. For these purposes, autologous transplantation models employing transgenic rats and wild-type rats, which were genetically identical to each other except for transgenes, were used. Two transplantation models were designed so that either the graft or the host cells had transgenes. Model I: transgenic rats were the donor, and wild-type rats were the recipient; model II: conversely, wild-type rats were the donor, and transgenic rats were the recipient. The grafted bone marrow cells and osteocytes in the trabeculae survived in the graft at 3 weeks after transplantation. Invasion of the host bone marrow cells into the graft was also found. Thus, bone marrow cells in the host as well as both bone marrow cells and osteocytes in the graft could potentially participate in the remodeling of underlying subchondral bone. Furthermore, the interface between graft and host was consisted with both graft and host derived cells. Since new bone formation was found in this space, both graft and host cells could have the potential to contribute to remodeling of underlying subchondral bone. The two models of the transplantations using the transgenic rats were found to be beneficial in following graft cells as well as host cells and in understanding their function on healing after autologous transplantation.     

磁共振显微线圈高分辨成像技术在腕关节病变中的临床应用

目的探讨磁共振显微线圈高分辨成像技术在腕关节病变中的临床应用。方法收集2018年12月至2019年12月45例腕关节类风湿关节炎患者、30例手腕关节骨性关节炎患者、40例腕部撞击综合症损伤患者作为研究对象,分别使用磁共振常规线圈及显微线圈技术对其部位进行扫描。比较分析两种诊断方式的诊断准确率及对软骨侵蚀、关节积液和滑膜增厚的诊出情况。结果显微线圈技术对腕关节类风湿关节炎、手腕关节骨性关节炎、腕部撞击综合症损伤的诊断准确率分别为97.78%、96.67%、97.50%,均显著高于常规线圈技术的60.00%、50.00%、60.00%,差异有统计学意义(P<0.05)。显微线圈技术对软骨侵蚀、关节积液和滑膜增厚的诊出情况显著优于常规线圈技术,差异有统计学意义(P<0.05)。经显微线圈技术MRI增强扫描检测:腕关节类风湿关节炎患者右手近端指间关节周围软组织增厚,关节周围可见少量积液,增强扫描指间关节滑膜呈明显增厚并强化。骨性关节炎患者可见关节面下骨质异常信号,表现为“虫蚀状”或小斑片状软骨下骨质缺损区。结论磁共振显微线圈高分辨成像技术能够显著提高腕关节病变的诊断准确率,提高对软骨侵蚀、关节积液和滑膜增厚的诊出情况,值得广泛推广。     

同轴骨组织工程支架降解特性研究

为使抗骨结核药物在病灶区长期维持一定浓度,并促进术后缺损处的骨修复,应用羟基磷灰石、聚乙烯醇、丝素蛋白作为药物载体材料,结合自制机械式挤出装置制备同轴梯度骨组织工程支架.为使骨组织工程支架顺利植入体内,且保证支架负载的药物持续缓慢地释放,现对药物载体材料骨组织工程支架降解性能进行测试.研究发现降解10周后,无丝素蛋白的...     

PLLA/CPC复合支架增强结构设计及制备

为制备用于负重部位骨缺损修复的人工植入体,提出了一种复合增强结构人工骨设计及制备方案,该方案采用聚左旋乳酸支架(PLLA)为增强结构,以自凝固磷酸钙骨水泥(CPC)为复合填充材料,制备了复合增强结构人工骨。研究了PLLA的棒材力学性能、支架设计和制作及其不同增强结构形式对人工骨力学性能的影响。力学试验结果表明,所制作的人工骨有效提高了承载能力,并且该复合结构能有效弥补金属骨无法降解的缺陷。     

Influence of surgical preparation on the in-vivo response of osteochondral defects

Cartilage has an extremely poor capacity to heal, which has lead to intensive research into biomaterials and tissue engineering for the purpose of regenerating cartilage in vivo. Many of these techniques have shown great promise in vitro; however, the results do not always carry across to the in-vivo scenario. Healthy cartilage autografts often do not integrate with the adjacent cartilage, suggesting that cartilage is rarely capable of healing even under ideal conditions. It is hypothesized in this study that the surgical creation of defects in cartilage causes significant damage to the adjacent tissues, leading to further degradation of the cartilage and poor outcome for the repair in general. This study compares the healing response of osteochondral defects created with either a punch or a drill in the weight-bearing region of the sheep knee at 4 and 26 weeks following surgery. The use of a drill to create the defect creates a more aggressive inflammatory response at 4 weeks compared with a punch. However, by 26 weeks, defects created with a punch scored higher on the O'Driscoll cartilage grading scale. Tissue damage at the time of surgery plays an important part in the sequence of events for healing of cartilage defects. This knowledge will help to characterize and refine the ovine model for cartilage regeneration and may have an influence on surgical technique and instrumentation for clinical cartilage repair.     

Stem cells for tissue engineering of articular cartilage

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.     

Mechanical properties and in-vivo performance of calcium phosphate cement-chitosan fibre composite

Self-hardened calcium phosphate cement (CPC) sets to form hydroxyapatite and possesses excellent osteoconductivity. However, lack of macroporosity and low strength constrain its application in bone tissue engineering. Recent studies have incorporated various fibres into CPC to improve its mechanical strength. The present approach focused on the reinforcement of CPC with chitosan fibres and then the effects of the fibre structure on the mechanical properties and macrochannels formation characteristics of CPC-fibre composite were investigated. Chitosan fibres of diameter 200 microm were used to fabricate two types of three-dimensional structure, which were then coated with collagen and incorporated into CPC to fabricate CPC-fibre implants with a fibre volume content of 5 per cent. The compressive strength of the CPC-fibre implant was 33 MPa when the strain was 2.4 per cent, which is fourfold higher than that of the CPC control. Nine cylindrical implants including six CPC-fibre implants were implanted in the bone defects of nine dogs and were then post-operatively observed. After 20 weeks in vivo, new callus from the healthy tissue of the defect entirely integrated with the CPC-fibre implant and new bone was formed as the implant degraded. Scanning electronic microscopy images indicated that macrochannels were formed in the CPC-fibre implants with the degradation of fibres, but only micropores with a scale of less than 50 microm could be observed in the CPC control. Briefly, the incorporation of a suitable chitosan-fibre structure into a CPC implant not only could improve its mechanical properties but also facilitated the bone repair process in vivo.     

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.     

Fabrication of porous beta-tricalcium phosphate with microchannel and customized geometry based on gel-casting and rapid prototyping

The tissue engineering scaffolds with three-dimensional porous structure are regarded to be beneficial to facilitate a sufficient supply of nutrients and enable cell ingrowth in bone reconstruction. However, the pores in scaffolds tend to be blocked by the cell ingrowth and result in a restraint of nutrient supply in the further side of the scaffold. An indirect approach of combining the rapid prototyping and gel-casting technique is introduced in this study to fabricate beta-tricalcium phosphate (beta-TCP) scaffolds which not only have interconnected porous structure, but also have a microchannel network inside. The scaffold was designed with customized geometry that matches the defect area, and a double-scale (micropores-microchannel) porous structure inside that is beneficial for cell ingrowth. The scaffolds fabricated have an open, uniform, and interconnected porous architecture with a pore size of 200-400 microm, and posses an internal channel network with a diameter of 600 microm. The porosity was controllable. The compressive yield strength was 4.5 MPa with a porosity of 70 per cent. X-ray diffraction analysis shows that these fabrication processes do not change the crystal structure and chemical composition of beta-TCP. With this technique, it was also possible to fabricate porous scaffolds with desired pore size, porosity, and microchannel, as well as customized geometries by other bioceramics.     

Traceability needs for geometrical identification of form of biobearings working surfaces and defect of joint cartilage

In this paper the own system for traceability analysis of geometrical estimation of the articulation cartilage defects imaged using magnetic resonance (MR) is presented. There were examined three animals and two human specimen knees using MR and coordinate control machine (CCM). Three dimension modeling and visualization of the defect using finite elements method in the packets were done: Ansys, FEMAP, NE Nastran. Computer tomography was done to verify bone damage. The size of the cartilage defect obtained in MR and CCM were approximately. MR and CCM are the sufficient methods of traceability for imaging of cartilage defects. The quality of the shape imagine and size estimation of the defects depends on the measurement strategy and selection of the best imaging sequences. The described procedure of the MR application to the modeling computed programs like FEMAP, NE Nastran will give the possibility for creation of the virtual imaging of the real articulation cartilage defects. The described procedure of cartilage defects diagnosis can be used as a modern, non-invasion method for preoperative planning of way of treatment.     

The fate of implanted autologous chondrocytes in regenerated articular cartilage

Autologous chondrocyte implantation (ACI) is used to treat some articular cartilage defects. However, the fate of the cultured chondrocytes after in-vivo transplantation and their role in cartilage regeneration remains unclear. To monitor the survival and fate of such cells in vivo, the chondrocytes were labelled with a lipophilic dye and the resultant regenerated tissue in dogs examined. It was found that, 4 weeks after implantation, the osteochondral defects were filled with regenerative tissue that resembled hyaline cartilage. Fluorescence microscopy of frozen sections of the regenerated tissue revealed that the majority of cells were derived from the DiI-labelled implanted chondrocytes. From these results, it was concluded that a large population of implanted autologous chondrocytes can survive at least 4 weeks after implantation and play a direct role in cartilage regeneration. However, it remains unknown whether other cells, such as periosteal cells or bone marrow stromal stem cells, are involved in the regeneration of cartilage after ACI.     

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.     

Autologous resurfacing

Background: There is a discrepancy between the interest in joint‐reconstructions and the current knowledge about the healing‐processes involved. Major reconstructions are performed with osteosynthesized allografts and fresh allografts for cartilage. Objectives: The main question to be answered is: what do we know about metaphyseal and epiphyseal cancellous bone healing, contact healing of the subchondral bone and its influence on cartilage healing? Can we achieve healing of all four compartments in contact? Purpose: The purpose is to systematically investigate through animal testing the healing processes of metaphyseal and epiphyseal bone, including the subchondral bone and the healing of cartilage of press‐fit‐inserted grafts, considering nondemineralized high‐resolution histology. Material and Methods: Primary cancellous‐bone healing of osteosynthesized hemi‐osteotomies was studied in 13 canine tibial heads, the contact healing was investigated in 7 dogs and 18 giant‐rabbits comparing contact‐healing of press‐fit‐inserted autologs cylindrical grafts with empty defects applying the wet‐grinding diamond‐technology. Bench‐experiments on the epiphyseal bones of swine including pullout‐tests of cylindrical grafts formed the basis for validation of that press‐fit diamond technology. Results: Primary metaphyseal and epiphyseal contact healing, including hyaline cartilage, was found in all compartments of the meta‐and epiphysis in the precisely performed experiments. The press‐fit principle, which employs cylindrical grafts and diamond instrumentation featuring a difference of 15/100 mm between graft and recipient bed, achieved high loads between 73.48 and 178.95 N (mean value 118.16 and standard deviation 32.79) in the pullout tests. Conclusion: Autologous press‐fit grafting with alignment of the bony baseplate using wet‐grinding precision has attained promising histo‐morphological results. Microsc. Res. Tech., 78:40–51, 2015. © 2014 Wiley Periodicals, Inc.     

Characterization of compressive deformation behavior of multi-layer porous composite materials for articular tissue engineering

Regeneration of articular layered tissues consisting of cartilage and cancellous bone has been a critical issue in orthopedics. Tissue engineering technology for such large-scale damaged layered tissue may be developed by using layered scaffold with stem cells. In this study, therefore, a novel multi-layer scaffold consisting of a porous poly (?-caprolactone) (PCL) layer for cartilage regeneration and a porous composite layer of poly (L-lactic acid) (PLLA) and hydroxyapatite (HAp) for bone regeneration was developed. The microstructure of the scaffold was characterized by a field emission scanning electron microscope (FE-SEM). Compression tests were also performed to understand the stress-strain behavior. FE-SEM observation clearly showed that an interlayer exists between the PCL and the composite layers. The compressive stress-strain relation is characterized by a stepwise behavior including the first and the second steps. The first modulus corresponding to the first step is mainly related to the deformation of the PCL layer; on the other hand, the second modulus is related to both solidified PCL layer and the composite layer and increases with increase of HAp content of the composite layer. It is also found that the classical mechanics theory and three-dimensional finite element model can predict the first modulus reasonably well.     

Bone repair investigation using rhBMP-2 and angiogenic protein extracted from latex

Background: The aim of this work was to study the new bone tissue formation after bone morphogenetic protein type 2 (rhBMP‐2) and P‐1 application, using 5 and 10 μg of each, combined to a material carrier, in critical bone defects. Methods: It was used 70 Wistar rats (male, ～250 g) that were divided in 10 groups with seven animals on each. Groups are the following: critical bone defect only, pure monoolein gel, 5 μg of pure P‐1, 5 μg of pure rhBMP‐2, 5 μg of P‐1/monoolein gel, 5 μg of rhBMP‐2/monoolein gel, 10 μg of pure P‐1, 10 μg of pure rhBMP‐2, 10 μg of P‐1/monoolein gel, 10 μg of rhBMP‐2/monoolein gel. Animals were sacrificed after 4 weeks of the surgical procedure and the bone samples were submitted to histological, histomorphometrical, and immunohistochemical evaluations. Results: Animals treated with pure P‐1 protein, in both situations with 5 μg and 10 μg, had no significant difference (P > 0.05) for new bone formation; other groups treated with 10 μg were statistically significant (P < 0.05) among themselves and when compared with groups in which it was inserted the monoolein gel or critical bone defect only (P < 0.05). In the group involving the 10 μg rhBMP‐2/monoolein gel association, it was observed an extensive bone formation, even when compared with the same treatment without the gel carrier. Conclusion: Using this experimental animal model, more new bone tissue was found when it was inserted the rhBMP‐2, especially when this protein was combined to the vehicle, and this process seems to be dose dependent. Microsc. Res. Tech., 2011.© 2011 Wiley Periodicals, Inc.     

Oral tissue response to ovine grafting biomaterial: Morphological and morphometric study using scanning electron and light microscopy tissue response to ovine grafting biomaterial

OBJECTIVE: To evaluate the oral tissue response to an experimental particle ovine biomaterial by scanning electron microscopy (SEM) and light microscopy (LM). MATERIAL AND METHODS: Forty‐eight rats had surgical periodontal defects treated with either blood clotting (control), bovine biomaterial? (B), or an experimental ovine biomaterial (O). Data from SEM analysis (defect exposure, root surface exposure, diameter of matrix fibers and bundles, and globuli areas; n = 5) were applied to Shapiro–Wilk, Kruskal–Wallis, and Dunn's test, whereas LM analysis (tissue cicatrization characteristics and diameter defect; n = 3) had data applied to two‐way analysis of variance. Animals were monitored for 1 and 3 weeks. RESULTS: By SEM, the O samples showed significant differences from B and C in the area of defect exposure (H_2,15 = 8.66; P < 0.05). In both periods, O and B samples showed similar results for matrix fiber diameters, differently than C samples (H_2,15 = 14.0; P < 0.05). All other SEM variables were considered equivalent among the groups (P > 0.05). Under LM, an acute and chronic granulomatous inflammation was seen in the presence of both biomaterials (B and O, 1 week); both the control and the ovine grafting samples showed mature bone in the repair site (3 weeks); the defect diameter showed similar values among groups, at both monitoring periods (F_2,12 = 1.0401; P > 0.05). CONCLUSION: The ovine particles of this study showed a favorable response to oral tissue repair, demonstrating to be a potential source for the development of bone grafting biomaterials. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.