Nerve regeneration with aid of nanotechnology and cellular engineering

Repairing nerve defects with large gaps remains one of the most operative challenges for surgeons. Incomplete recovery from peripheral nerve injuries can produce a diversity of negative outcomes, including numbness, impairment of sensory or motor function, possibility of developing chronic pain, and devastating permanent disability. In the last few years, numerous microsurgical techniques, such as coaptation, nerve autograft, and different biological or polymeric nerve conduits, have been developed to reconstruct a long segment of damaged peripheral nerve. A few of these techniques are promising and have become popular among surgeons. Advancements in the field of tissue engineering have led to development of synthetic nerve conduits as an alternative for the nerve autograft technique, which is the current practice to bridge nerve defects with gaps larger than 30 mm. However, to date, despite significant progress in this field, no material has been found to be an ideal alternative to the nerve autograft. This article briefly reviews major up-to-date published studies using different materials as an alternative to the nerve autograft to bridge peripheral nerve gaps in an attempt to assess their ability to support and enhance nerve regeneration and their prospective drawbacks, and also highlights the promising hope for nerve regeneration with the next generation of nerve conduits, which has been significantly enhanced with the tissue engineering approach, especially with the aid of nanotechnology in development of the three-dimensional scaffold. The goal is to determine potential alternatives for nerve regeneration and repair that are simply and directly applicable in clinical conditions.     

Drug releasing systems in cardiovascular tissue engineering

Heart disease and atherosclerosis are the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts; however, current artificial grafts carry significant limitations, including thrombosis, infection, limited durability and the inability to grow. Tissue engineering of blood vessels, cardiovascular structures and whole organs is a promising approach for creating replacement tissues to repair congenital defects and/or diseased tissues. In an attempt to surmount the shortcomings of artificial grafts, tissue-engineered cardiovascular graft (TECVG), constructs obtained using cultured autologous vascular cells seeded onto a synthetic biodegradable polymer scaffold, have been developed. Autologous TECVGs have the potential advantages of growth, durability, resistance to infection, and freedom from problems of rejection, thrombogenicity and donor scarcity. Moreover polymers engrafted with growth factors, cytokines, drugs have been developed allowing drug-releasing systems capable of focused and localized delivery of molecules depending on the environmental requirements and the milieu in which the scaffold is placed. A broad range of applications for compound-releasing, tissue-engineered grafts have been suggested ranging from drug delivery to gene therapy. This review will describe advances in the development of drug-delivery systems for cardiovascular applications focusing on the manufacturing techniques and on the compounds delivered by these systems to date.     

Incorporation of Chitosan Microspheres into Collagen-Chitosan Scaffolds for the Controlled Release of Nerve Growth Factor

Background

Artifical nerve scaffold can be used as a promising alternative to autologous nerve grafts to enhance the repair of peripheral nerve defects. However, current nerve scaffolds lack efficient microstructure and neurotrophic support.

Methods

Microsphere–Scaffold composite was developed by incorporating chitosan microspheres loaded with nerve growth factor (NGF–CMSs) into collagen-chitosan scaffolds (CCH) with longitudinally oriented microchannels (NGF–CMSs/CCH). The morphological characterizations, in vitro release kinetics study, neurite outgrowth assay, and bioactivity assay were evaluated. After that, a 15-mm-long sciatic nerve gap in rats was bridged by the NGF–CMSs/CCH, CCH physically absorbed NGF (NGF/CCH), CCH or nerve autograft. 16 weeks after implantation, electrophysiology, fluoro-gold retrograde tracing, and nerve morphometry were performed.

Results

The NGF–CMSs were evenly distributed throughout the longitudinally oriented microchannels of the scaffold. The NGF–CMSs/CCH was capable of sustained release of bioactive NGF within 28 days as compared with others in vitro. In vivo animal study demonstrated that the outcomes of NGF–CMSs/CCH were better than those of NGF/CCH or CCH.

Conclusion

Our findings suggest that incorporation of NGF–CMSs into the CCH may be a promising tool in the repair of peripheral nerve defects.     

Feasibility of repairing skin defects by VEGF165 gene-modified iPS-HFSCs seeded on a 3D printed scaffold containing astragalus polysaccharide

The preparation of biodegradable scaffolds loaded with cells and cytokine is a feature of tissue-engineered skin. IPSCs-based tissue-engineered skin treatment for wound repair is worth exploring. Healthy human skin fibroblasts were collected and reprogrammed into iPSCs. After gene modification and induction, CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP were obtained and identified by a combination of immunofluorescence and RT-qPCR. Astragalus polysaccharide-containing 3D printed degradable scaffolds were prepared and co-cultured with VEGF₁₆₅ gene-modified iPS-HFSCs^GFP, and the biocompatibility and spatial structure of the tissue-engineered skin was analysed by cell counting kit-8 (CCK8) assay and scanning electron microscopy. Finally, the tissue-engineered skin was transplanted onto the dorsal trauma of nude mice, and the effect of tissue-engineered skin on the regenerative repair of total skin defects was evaluated by a combination of histology, immunohistochemistry, immunofluorescence, RT-qPCR, and in vivo three-dimensional reconstruction under two-photon microscopy. CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP, close to the morphology and phenotype of human-derived hair follicle stem cells, were obtained. The surface of the prepared 3D printed degradable scaffold containing 200 μg/mL astragalus polysaccharide was enriched with honeycomb-like meshwork, which was more conducive to the proliferation of the resulting cells. After tissue-engineered skin transplantation, combined assays showed that it promoted early vascularization, collagen and hair follicle regeneration and accelerated wound repair. VEGF₁₆₅ gene-modified iPS-HFSCs^GFP compounded with 3D printed degradable scaffolds containing 200 μg/mL astragalus polysaccharide can directly and indirectly participate in vascular, collagen, and hair follicle regeneration in the skin, achieving more complete structural and functional skin regenerative repair.     

Transdifferentiated Mesenchymal Stem Cells as Alternative Therapy in Supporting Nerve Regeneration and Myelination

1. Aims: Demyelination plays a crucial role in neurodegenerative processes and traumatic disorders. One possibility to achieve remyelination and subsequent restoration of neuronal function is to provide an exogenous source of myelinating cells via transplantation. In this context, mesenchymal stem cells (MSCs) have attracted interest. They are multipotent stem cells that differentiate into cells of the mesodermal lineage like bone, cartilage, fat, and muscle. Although adult, their differentiation potential is remarkable, and they are able to transdifferentiate.2. Methods: We transformed cultivated rat MSCs into myelinating cells by using a cytokine cocktail. Transdifferentiated MSCs were characterized by an enhanced expression of LNGF-receptor, Krox20, and CD104, and a decreased expression of BMP receptor-1A as compared to untreated MSCs. The myelinating capacity was evaluated in vitro and in vivo. Therefore, PC12 cells, normally unmyelinated, were cocultivated with MSCs, transdifferentiated MSCs, and Schwann cells, or the respective cells were grafted into an autologous muscle conduit bridging a 2-cm gap in the rat sciatic nerve. Myelination of PC12 cells was demonstrated by electron microscopy. In vivo, after 3 and 6 weeks regeneration including myelination was monitored histologically and morphometrically. Autologous nerves and cell-free muscle grafts were used as control.3. Results: Schwann cells and transdifferentiated MSCs were able to myelinate PC12 cells after 14 days in vitro. In vivo, autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate myelination was noted in the Schwann cell groups and, albeit with restrictions, in the transdifferentiated MSC groups, while regeneration in the MSC groups and in the cell-free groups was impaired.4. Conclusion: Our findings demonstrate that it may be possible to differentiate MSCs into therapeutically useful cells for clinical applications in myelin defects.     

脂肪干细胞在周围神经损伤修复中的作用

周围神经损伤的修复是临床外科中的一个难题。尽管周围神经系统在损伤后具有内在的自我修复能力,但一般很难达到完全功能恢复,特别是近端的损伤或者大段的神经缺损。近年来,基于干细胞的细胞治疗为周围神经再生带来了曙光。大量研究表明干细胞可促进周围神经损伤的再生,然而其作用机制还不明确。为此,本文将对脂肪干细胞在周围神经损伤修复中作用包括向雪旺细胞分化、神经营养、血管形成、神经元保护、靶器官保护和免疫调节等作用进行归纳,并进一步探讨其潜在的作用机制。     

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

Dihydrotestosterone Treatment Accelerates Autograft Reversal Sciatic Nerve Regeneration in Rats

Neuroactive steroids such as progesterone, testosterone, and their derivatives have been widely studied for their neuroprotective roles in the nervous system. Autologous nerve transplantation is considered as the gold standard repair technique when primary suture is impossible; nevertheless, this method is far from ideal. In this study, we aimed to explore the impact of dihydrotestosterone (DHT), a 5α-reduced derivative of testosterone, on the recovery of peripheral nerve injury treated with autologous nerve transplantation. Sprague–Dawley rats were subjected to a 10-mm right side sciatic nerve reversed autologous nerve transplantation and randomly divided into groups that received DHT or DHT?+?flutamide (an androgen receptor blocker) daily for 8 weeks after operation. Our results demonstrated that DHT could speed up the rate of axonal regeneration and increase the expression of myelin protein zero (P0) in autograft reversal sciatic nerves. Thus, our study provided new insights into improving the prognosis of patients with long gap peripheral nerve defects.     

电刺激联合干细胞修复周围神经损伤的研究进展

周围神经损伤在创伤中较为常见,易造成神经系统部分或全部损伤,从而导致功能丧失和其他神经性疾病．尽管周围神经损伤的治疗效果随着科技的发展有了明显提高,但距离真正的形态和功能重建还相差甚远,神经再生及功能恢复速度缓慢仍是临床治疗的难点．电刺激因使用方便、无创和副作用小等优点越来越受到研究者的青睐,与干细胞联合广泛用于周围神经损伤修复的体外研究．本文论述了电刺激联合干细胞在周围神经损伤修复方面的研究进展,并讨论了其可能的作用机理．特别分析了电刺激联合干细胞在周围神经损伤修复研究中的难点,展望了其发展前景．     

Human amniotic fluid stem cells attract osteoprogenitor cells in bone healing

Current treatments of large bone defects are based on autologous or allogenic bone transplantation. Human amniotic fluid stem cells (hAFSCs) were evaluated for their potential in bone regenerative medicine. In this study, hAFSCs were transduced with lentiviral vector harboring red fluorescent protein to investigate their role in the regeneration of critical-size bone defects in calvarial mouse model. To distinguish donor versus recipient cells, a transgenic mouse model carrying GFP fluorescent reporter was used as recipient to follow the fate of hAFSCs transplanted in vivo into Healos® scaffold. Our results showed that transduced hAFSCs can be tracked in vivo directly at the site of transplantation. The presence of GFP positive cells in the scaffold at 3 and 6 weeks after transplantation indicates that donor hAFSCs can recruit host cells during the repair process. These observations help clarify the role of hAFSCs in bone tissue repair.     

Role of Non-coding RNAs in Axon Regeneration after Peripheral Nerve Injury

Peripheral nerve injury (PNI) may lead to disability and neuropathic pain, which constitutes a substantial economic burden to patients and society. It was found that the peripheral nervous system (PNS) has the ability to regenerate after injury due to a permissive microenvironment mainly provided by Schwann cells (SCs) and the intrinsic growth capacity of neurons; however, the results of injury repair are not always satisfactory. Effective, long-distance axon regeneration after PNI is achieved by precise regulation of gene expression. Numerous studies have shown that in the process of peripheral nerve damage and repair, differential expression of non-coding RNAs (ncRNAs) significantly affects axon regeneration, especially expression of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In the present article, we review the cellular and molecular mechanisms of axon regeneration after PNI, and analyze the roles of these ncRNAs in nerve repair. In addition, we discuss the characteristics and functions of these ncRNAs. Finally, we provide a thorough perspective on the functional mechanisms of ncRNAs in nervous injury repair, and explore the potential these ncRNAs offer as targets of nerve injury treatment.     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.     

Conserved Dopamine Neurotrophic Factor-Transduced Mesenchymal Stem Cells Promote Axon Regeneration and Functional Recovery of Injured Sciatic Nerve

Peripheral nerve injury (PNI) is a common disease that often results in axonal degeneration and the loss of neurons, ultimately leading to limited nerve regeneration and severe functional impairment. Currently, there are no effective treatments for PNI. In the present study, we transduced conserved dopamine neurotrophic factor (CDNF) into mesenchymal stem cells (MSCs) in collagen tubes to investigate their regenerative effects on rat peripheral nerves in an in vivo transection model. Scanning electron microscopy of the collagen tubes demonstrated their ability to be resorbed in vivo. We observed notable overexpression of the CDNF protein in the distal sciatic nerve after application of CDNF-MSCs. Quantitative analysis of neurofilament 200 (NF200) and S100 immunohistochemistry showed significant enhancement of axonal and Schwann cell regeneration in the group receiving CDNF-MSCs (CDNF-MSCs group) compared with the control groups. Myelination thickness, axon diameter and the axon-to fiber diameter ratio (G-ratio) were significantly higher in the CDNF-MSCs group at 8 and 12 weeks after nerve transection surgery. After surgery, the sciatic functional index, target muscle weight, wet weight ratio of gastrocnemius muscle and horseradish peroxidase (HRP) tracing demonstrated functional recovery. Light and electron microscopy confirmed successful regeneration of the sciatic nerve. The greater numbers of HRP-labeled neuron cell bodies and increased sciatic nerve index values (SFI) in the CDNF-MSCs group suggest that CDNF exerts neuroprotective effects in vivo. We also observed higher target muscle weights and a significant improvement in muscle atrophism in the CDNF-MSCs group. Collectively, these findings indicate that CDNF gene therapy delivered by MSCs is capable of promoting nerve regeneration and functional recovery, likely because of the significant neuroprotective and neurotrophic effects of CDNF and the superior environment offered by MSCs and collagen tubes.     

Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep

Background

Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/Principal Findings

The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/Significance

This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.     

神经生长因子与冻干异体神经桥接大鼠神经缺损的研究

实验采用冻干处理的异体神经与外源性神经生长因子（ＮＧＦ）结合来桥接大鼠的坐骨神经１．０ｃｍ的缺损。用雄性Ｗｉｓｔａｒ大鼠进行的四组实验结果表明：冻干处理的异体神经可降低其抗原性,但处理后并不损害雪旺氏细胞（ＳＣ）基底膜的完整性,在移植后可能成为轴突再生的通道和支架;外源性ＮＧＦ与冻干神经结合形成的复合体,可为神经的再生提供一个较好的微环境,具有成为理想桥接材料的可能性     

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.     

Preliminary study on nerve regeneration through an autologous venous tube

Large posttraumatic defects in the peripheral nervous system bring up serious problems to the surgeon. There is no definitive answer yet. The authors present a model for nervous regeneration studies; venous autograft was used to bridge defects in a divided rat sciatic nerve; the venous tube guided the regenerating axons towards the distal stump over up to 1.9 cm length. Preliminary studies about nervous regeneration stimulation with autologous Schwann cells are discussed.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

干细胞治疗周围神经损伤研究进展

周围神经损伤是临床常见的疾病。损伤后神经的修复和再生是复杂又漫长的过程。严重的神经损伤其预后效果并不令人满意,相应支配区域的功能难以恢复,这给患者及家人带来了极大的痛苦。因此如何更好的对周围神经损伤进行治疗一直是医学界的难题。在神经修复机制的研究中,科学家发现施万细胞对周围神经的修复和再生起到了非常重要的作用,但获取和扩增的困难限制了其临床的应用。随着生物医学的发展,人们把目光投向了干细胞,经实验发现干细胞不仅具有旺盛的增殖能力,而且可以分化为神经系细胞,还能分泌相关的神经营养因子促进神经的修复和再生,这为周围神经损伤后的治疗带来了新的希望。本文就近些年来应用于修复周围神经的干细胞及促进修复机制的研究做以综述。