The role and effectiveness of bone marrow in osseous regeneration

Autologous bone marrow serves as the richest and most readily available repository of progenitor cells capable of differentiating into mature bone forming cells. Numerous studies have demonstrated that bone marrow alone or cell suspensions of marrow usually diluted in culture medium affect de novo bone formation when implanted in soft tissue sites. Consequently, when bone marrow is used to enrich orthopaedic grafting matrices such as xenograft, demineralized bone matrix, or ceramic materials it almost invariably produces faster and more consistent defect healing compared to bone marrow or the carrier matrix alone and, in some cases, equivalent healing to autograft. This article evaluates the clinical effectiveness of these grafting materials for various orthopaedic applications such as healing long bone defects and enhancing spinal fusion procedures, and hypothesizes that enrichment with bone marrow is integral to timely and satisfactory graft incorporation. Methods such as centrifugation, culture expansion and selective cell retention that concentrate and deliver marrow-derived osteoprogenitor cells to the graft site as a means of mimicking more closely the consistent bone forming potential of autologous bone graft are also discussed.     

Laser-induced regeneration of cartilage

Laser radiation provides a means to control the fields of temperature and thermo mechanical stress, mass transfer, and modification of fine structure of the cartilage matrix. The aim of this outlook paper is to review physical and biological aspects of laser-induced regeneration of cartilage and to discuss the possibilities and prospects of its clinical applications. The problems and the pathways of tissue regeneration, the types and features of cartilage will be introduced first. Then we will review various actual and prospective approaches for cartilage repair; consider possible mechanisms of laser-induced regeneration. Finally, we present the results in laser regeneration of joints and spine disks cartilages and discuss some future applications of lasers in regenerative medicine.     

Structure, formation and role of cartilage canals in the developing bone

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.     

骨膜在骨折愈合及骨组织修复过程中的作用

背景:骨膜因含有成骨潜能的细胞而参与骨折愈合及骨组织修复的过程,其在修复骨缺损方面的作用是目前研究的热点。目的:对骨膜在骨折愈合及骨组织修复过程中所发挥的作用进行综述。方法:第一作者应用计算机检索1964年1月至2019年12月PubMed、中国知网数据库等中英文期刊全文数据库有关骨膜在骨折愈合及骨组织修复过程中作用的文章,英文检索词periosteum,bone healing,periosteum cells;中文检索词骨膜,骨折愈合,骨膜细胞。最终选择48篇重要文献进行综述。结果与结论:①骨膜的完整性对于骨折愈合和骨组织的修复极其重要,因其较好的成骨性能、优质的材料学特性和屏障作用等特点被广泛应用于骨组织工程;②骨膜在牵拉成骨过程中的作用机制目前尚且存在争议,研究证实骨膜缺如并不会影响下颌骨缺损牵拉成骨的效果,但在四肢骨缺损牵拉成骨中的作用仍有待进一步研究。     

国内带血管蒂骨、骨膜瓣移位术近况

<正>四肢骨缺损、骨不连和骨缺血性坏死等病损的治疗历来是骨科医生十分关注的课题。上世纪70年代中期,由于显微外科技术的应用已使这一难题的解决初现曙光。在早期阶段,因仅有少量供区可供选择,所采用的多为吻合血管的骨或骨膜瓣游离移植,而游离组织瓣移植则存在着手术创伤大、费时长和难度较高等不足之处。     

The role of trabecular demineralized bone in combination with perichondrium in the generation of cartilage grafts

The use of a composite graft of bovine trabecular demineralized bone matrix (DBM) and perichondrium has been found a reliable method for in vivo generation of cartilage. In the present study, the mechanism whereby this commercially available matrix increases cartilage formation was investigated. First, the time course of cartilage formation in vivo, in the combined implant of perichondrium and DBM in the rabbit ear was studied, with special focus on tissue reactions to DBM. DBM was colonized by macrophages from day 3 post-operatively, reaching a maximum after 2 weeks. Only a minimal number of neutrophils was found. After 3 weeks the DBM appeared to be resorbed. In the first week the DBM was invaded with chondroblasts, and chondrogenesis occurred between the first and second week of implantation. After 3 weeks, the initially formed islets of cartilage had fused. Next, the chondrogenic capacity of DBM itself was investigated by implantation of DBM without perichondrium. This never resulted in cartilage formation. Immunohistochemistry showed only a faint staining of the DBM for growth factors. This indicates a minimal chondrogenic effect of DBM alone and the requirement of perichondrium as cell provider. In order to define the conditions which cause chondrogenesis in composites of perichondrium and DBM, a series of in vitro culture experiments was performed in which the in vivo situation was mimicked step by step. The basic condition was perichondrium cultured in medium with 10% FCS. In this condition, cartilage formation was variable. Because in the in vivo situation both DBM and macrophages can release growth factors, the effect of IGF1, TGFbeta2 or OP1 added to the culture medium was tested. Neither the incidence nor the amount of cartilage formation was stimulated by addition of growth factors. Perichondrium wrapped around DBM in vitro gave cartilage formation in the perichondrium but the incidence and amount were not significantly stimulated compared to cultures of perichondrium without DBM. However, cartilage-like cells were found in the DBM suggesting an effect of DBM on perichondrium-derived cells. Finally, macrophages and/or blood were added to the composite DBM-perichondrium to mimic the in vivo situation as close as possible. However, no effect of this treatment was found. In conclusion, this study indicates that DBM itself has few chondrogenic qualities but functions merely as a spacer for cell ingrowth. The fast resorption of DBM by macrophages in vivo seems of importance for the cartilage forming process, but in vitro the presence of macrophages (in combination with blood) could not enhance chondrogenesis.     

Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery

Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.     

Tracheal cartilage regeneration and new bone formation by slow release of bone morphogenetic protein (BMP)-2

We investigated the efficiency of bone morphogenetic protein (BMP)-2 released slowly from gelatin sponge for tracheal cartilage regeneration. A 1-cm gap was made in the mid-ventral portion of each of 10 consecutive tracheal cartilages. In the control group (n = 4), the resulting gap was left untreated. In the gelatin group (n = 4), plain gelatin was implanted in the gap. In the BMP-2 group (n = 4), gelatin containing 100 microg BMP-2 was implanted. We euthanatized all dogs in each group at 1, 3, 6, and 12 months after the implantation, respectively, and then examined the implant site macro- and microscopically. In the BMP-2 group, regenerated fibrous cartilage and newly formed bone were observed at 1 and 12 months. Regenerated cartilage was observed at the ends of the host cartilage stumps, with newly formed bone in the middle portion. The gaps were filled with regenerated cartilage and newly formed bone. At 3 and 6 months, regenerated cartilage, but not newly formed bone, was evident. The regenerated cartilage was covered with perichondrium and showed continuity with the host cartilage. We succeeded in inducing cartilage regeneration and new bone formation in canine trachea by slow release of 100 microg BMP-2 from gelatin.     

The role of proteoglycans in regeneration and plasticity

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated in glial scar tissue and inhibit axon regeneration. Not only are the protein cores up-regulated, but also there is more glycosaminoglycan (GAG) attached to them. The final stage of GAG synthesis is sulfation, which can occur in three positions. Both 6-sulfated GAG and the sulfotransferase that sulfates N -acetylgalactosamine in the 6 position is specifically up-regulated in glial scar tissue, in inhibitory glial cells and in astrocytes treated with tumour growth factor α (TGF-α) and TGF-β. Removal of GAG chains by digestion with chondroitinase or inhibition of GAG synthesis with chlorate or β-D-xylosides, removes much of the inhibition from CSPGs in vitro . We therefore tested to see whether GAG digestion by chondroitinase would promote axon regeneration in vivo . We first treated mechanical lesions of the nigrostriatal tract and saw regeneration of about 4% of axons back to their target. Next, dorsal column lesions of the spinal cord at C4 were treated. Both sensory and corticospinal axons regenerated in treated cords, and there was rapid return of function in beam and grid walking tests. The return of function was so rapid that we hypothesized that some of it might be due to enhanced plasticity. Many neuronal cell bodies and dendrites are coated in thick perineuronal nets of inhibitory CSPGs and tenascin-R, which would certainly be expected to prevent the formation of new synapses. We therefore tested the effects of chondroitinase treatment in a plasticity model: ocular dominance shift in the visual cortex following monocular deprivation. Monocular deprivation in adult animals normally produces no ocular dominance shift. However, in adult animals in which the cortex was treated with chondroitinase, there was a large shift in response to monocular deprivation.     

Innervation of human bone periosteum by peptidergic nerves

Nerves exhibiting substance P-like immunoreactivity were demonstrated in the human periosteum. A network of nerves showing substance P-like immunoreactivity was seen in the periosteum, while finer strands of immunoreactive nerve fibers were present immediately beneath the surface of the periosteum. Enkephalin-like immunoreactivity was also studied but could not be demonstrated. Substance P has previously been suggested to be involved in the mediation of the sensation of pain. The clinically observable marked pain sensitivity of periosteal tissue might be explained by the peptidergic nerves described in this paper.     

Comparative evaluation of MSCs from bone marrow and adipose tissue seeded in PRP-derived scaffold for cartilage regeneration

The aims of this study were to (1) determine whether platelet-rich plasma (PRP) could be prepared as a bioactive scaffold capable of endogenous growth factor release for cartilage repair; (2) compare the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) from bone marrow (BMSC) and from adipose (ADSC) seeded within the PRP scaffold; and (3) test the efficacy of ADSC-PRP construct in cartilage regeneration in?vivo. In?vitro evaluation showed that a 3-dimensional scaffold with a mesh-like microstructure was formed from PRP, with the capability of endogenous growth factor release and ready cell incorporation. Upon seeding in the PRP scaffold, BMSC showed higher proliferation rate, and higher expression of cartilage-specific genes and proteins than ADSC. In an osteochondral defect model in rabbits, implanted BMSC seeded within PRP scaffold also exhibited better gross appearance and histological and immunohistochemical characteristics, higher cartilage-specific gene and protein expression as well as subchondral bone regeneration. ADSC seeded constructs developed into functional chondrocytes secreting cartilaginous matrix in rabbits at 9 weeks post-implantation. Our findings suggest that PRP is a candidate bioactive scaffold capable of releasing endogenous growth factors and that BMSC and ADSC seeded within the PRP scaffold differentiate into chondrocytes and may be suitable for cell-based cartilage repair.     

The role of rhFGF-2 soaked polymer membrane for enhancement of guided bone regeneration

Abstract

The purposes of this study are to confirm the role of Fibroblast Growth Factor-2 (FGF-2) in bone regeneration by adding various concentrations of FGF-2 to the collagen membrane and applying it to the Biphasic Calcium Phosphate (BCP) bone graft site for guided bone regeneration, to explore the potential of collagen membrane as FGF-2 carrier, and to determine the optimum FGF concentration for enhancement of bone regeneration. Four bone defects of 8 mm in diameter were created in 18 New Zealand rabbit calvaria. After BCP bone graft, graft material was covered with collagen membranes adding various concentration of FGF-2. The concentration of FGF-2 was set at 1.0, 0.5, 0.1 mg/ml, and same amount of saline was used in the control group. To confirm the bone regeneration over time, six New Zealand rabbits were sacrificed each at 2, 4, and 12 weeks, and the amounts of new bone and residual bone graft material were analyzed by histologic and histomorphometric analysis. Qualitative analyses are also conducted through immunohistochemistry, Tetrate-resistant acid phosphatase (TRAP) stain and Russell-Movat pentachrome stain. As the healing period increased, the formation of new bone increased and the amount of residual graft material decreased in all experimental groups. Immunohistochemistry, TRAP staining and pentachrome staining further showed that the addition of FGF-2 promoted bone regeneration in all experimental groups. It was also confirmed that polymer collagen membrane can be used as a useful carrier of FGF-2 when enhanced early stage of new bone formation is required.     

The impact of PLGA scaffold orientation on in vitro cartilage regeneration

The success of in vitro cartilage regeneration provides a promising approach for cartilage repair. However, the currently engineered cartilage in vitro is unsatisfactory for clinical application due to non-homogeneous structure, inadequate thickness, and poor mechanical property. It has been widely reported that orientation of scaffolds can promote cell migration and thus probably contributes to improving tissue regeneration. This study explored the impact of microtubular oriented scaffold on in vitro cartilage regeneration. Porcine articular chondrocytes were seeded into microtubule-oriented PLGA scaffolds and non-oriented scaffolds respectively. A long-term in vitro culture followed by a long-term in vivo implantation was performed to evaluate the influence of scaffold orientation on cartilage regeneration. The current results showed that the oriented scaffolds could efficiently promote cell migration towards the inner region of the constructs. After 12 weeks of in vitro culture, the chondrocyte-scaffold constructs in the oriented group formed thicker cartilage with more homogeneous structure, stronger mechanical property, and higher cartilage matrix content compared to the non-oriented group. Furthermore, the in vitro engineered cartilage based on oriented scaffolds showed better cartilage formation in terms of size, wet weight, and homogeneity after 12-week in vivo implantation in nude mice. These results indicated that the longitudinal microtubular orientation of scaffolds can efficiently improve the structure and function of in vitro engineered cartilage.     

骨膜联合同种异体骨治疗兔骨缺损模型的实验研究

目的　通过实验动物研究观察骨膜联合同种异体骨治疗骨缺损的效果,探讨其可行性及修复机制。  方法　以新西兰大白兔为研究对象,分为3组,即实验组、对照组和空白组。通过X线、HE染色及荧光定量PCR方法,检测骨膜联合同种异体骨修复骨缺损的效果及其可能机制。  结果　X线检查显示实验组与对照组骨缺损处均已修复,空白组骨缺损处未修复;HE染色结果显示实验组新生骨小梁排列良好,仍有少量异体骨残留,对照组新生骨小梁排列整齐,空白组未见明显新生骨小梁;荧光定量PCR结果显示,术后实验组Spp-1、BMP-2基因表达高于对照组(P<0.05),ColⅠα1基因表达低于对照组(P<0.05)。  结论　骨膜联合同种异体骨能够有效地促进骨缺损的修复。     

The role of barrier membranes for guided bone regeneration and restoration of large bone defects: current experimental and clinical evidence

Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery. Although there are several methods for bone reconstruction, they all have specific indications and limitations. The concept of using barrier membranes for restoration of bone defects has been developed in an effort to simplify their treatment by offering a sinlge-staged procedure. Research on this field of bone regeneration is ongoing, with evidence being mainly attained from preclinical studies. The purpose of this review is to summarize the current experimental and clinical evidence on the use of barrier membranes for restoration of bone defects in maxillofacial and orthopedic surgery. Although there are a few promising preliminary human studies, before clinical applications can be recommended, future research should aim to establish the 'ideal' barrier membrane and delineate the need for additional bone grafting materials aiming to 'mimic' or even accelerate the normal process of bone formation. Reproducible results and long-term observations with barrier membranes in animal studies, and particularly in large animal models, are required as well as well-designed clinical studies to evaluate their safety, efficacy and cost-effectiveness.