Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel

This study investigates the ability of platelet-rich plasma (PRP) combined with biomaterials to enhance in vivo bone-repairing activity. A biodegradable hydrogel was prepared from gelatin, which has an affinity for various growth factors. Rabbit PRP was conventionally prepared by blood centrifugation and dropped onto freeze-dried gelatin hydrogel to obtain gelatin hydrogel incorporating PRP. Gelatin hydrogel incorporating PRP was applied to a bone defect of rabbit ulna to evaluate bone formation at the defect in terms of soft X-ray and histological examinations. As controls, fibrin incorporating PRP, empty gelatin hydrogel, and free PRP were applied to the defect; in addition, defect without any application was examined. Successful bone regeneration was observed at bone defect treated with gelatin hydrogel incorporating PRP, in marked contrast to the control groups. When in contact with gelatin, growth factors, such as platelet-derived growth factor and transforming growth factor beta(1), were released from the PRP. PRP growth factors are immobilized in the hydrogel through physicochemical interaction with gelatin molecules. The immobilized growth factors are released from the hydrogel in concert with hydrogel degradation. It is likely that the gelatin hydrogel permitted the controlled release of bioactive growth factors, resulting in factor-induced promotion of bone regeneration.     

富血小板血浆在软骨修复领域的研究进展

软骨是缺乏血运的组织,创伤后很难利用自身的间充质干细胞进行修复,难以自愈,持续的关节软骨缺损可引发局部疼痛、水肿反应以及功能障碍、运动受限等其他症状.以往的临床治疗方法包括骨髓刺激法、软骨膜移植、自体软骨细胞移植等,但效果有限.富血小板血浆(PRP)是自体血提取的血小板浓缩物,含有丰富的生长因子及其他组织再生必须的细胞因子,可为软骨细胞生存提供良好的微环境,有望成为促进软骨再生的新方法.     

富血小板血浆干预膝关节骨性关节炎模型兔关节软骨和滑膜的改变

文题释义： 富血小板血浆(PRP)：1993年Hood等首先提出富血小板血浆概念,并发现富血小板血浆含有丰富的血小板,其数目比全血中数目高3倍以上。血小板中含有大量的生长因子,如血小板衍生生长因子、转化生长因子β、类胰岛素生长因子、表皮生长因子、血管内皮生长因子等。 Ⅱ型胶原纤维(COL Ⅱ)：胶原纤维是关节软骨基质的重要组成结构之一,其中含量最多的Ⅱ型胶原纤维是构成软骨的基本框架,具有维持关节软骨的形态结构和抗张力强度的功能。基质金属蛋白酶为Ⅱ型胶原纤维的特异性降解酶,其中基质金属蛋白酶13降解Ⅱ型胶原纤维的速度是基质金属蛋白酶1的10倍。 背景：研究显示富血小板血浆具有很强的促进软骨细胞修复和增生作用。 目的：探讨富血小板血浆在骨性关节炎中对软骨细胞修复及滑膜炎症抑制的疗效。 方法：新西兰大白兔40只,于兔耳中央动脉取血后采用Hokugo等的方法制备富血小板血浆,同时检测外周血及富血小板血浆的血小板、血小板衍生生长因子、转化生长因子β和血管内皮生长因子水平。采用前交叉韧带切断法来制作动物模型后随机将兔分为实验组和对照组,实验组双侧膝关节每周注射1次0.3 mL 富血小板血浆;对照组每周注射1次0.3 mL无菌生理盐水,共注射10周。注射后第2,4,6,8,10周对兔进行大体观察及膝关节组织学观察;检测关节软骨Ⅱ型胶原蛋白及基质金属蛋白酶13水平,并进行软骨组织Mankin评分。实验方案经重庆医科大学动物实验伦理委员会批准。 结果与结论：①富血小板血浆中血小板、血小板衍生生长因子、转化生长因子β和血管内皮生长因子水平分别是正常血中的5.5,4.8,7.7和6.2倍(均P < 0.05);②注射后第6周实验组Mankin评分小于对照组(P < 0.05);③实验组第4,6,8,10周时Ⅱ型胶原蛋白水平明显高于对照组(P < 0.05);实验组第2,4,6,8,10周时基质金属蛋白酶13水平明显小于对照组(P < 0.05);④结果表明,关节腔内注射富血小板血浆能通过缓解关节滑膜炎症及延缓甚至阻断软骨细胞的损伤来抑制骨性关节炎的进展。 ORCID: 0000-0001-6301-4790(邱皓) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

富血小板血浆修复膝关节骨关节炎

BACKGROUND: Platelet-rich plasma (PRP) containing high levels of platelet-derived growth factor for knee osteoarthritis has achieved good clinical results; however, the effects of RPR on the repair of damaged articular cartilage are still controversial. OBJECTIVE: To study the effects of RPR on the repair of damaged articular cartilage in a rabbit model of knee osteoarthritis. METHODS:The model of osteoarthritis in the rabbit right knee was established by Hulth’s method. Autologous PRP (0.5 mL) (PRP group), sodium hyaluronate (0.5 mL) (sodium hyaluronate group), and normal saline (model group) were injected into the right knee joint cavity, respectively. The morphology of articular surface and nitric oxide contents in knee joint fluid were observed and determined at 8 weeks after surgery. RESULTS AND CONCLUSION: The morphology of articular cartilage in the PRP group was better than that in the other three groups. Mankin scores of articular cartilage and nitric oxide contents of knee joint fluid in the PRP group were significantly decreased compared with the model and sodium hyaluronate groups that in (P < 0.05), while increased compared with the sham-operation group (P < 0.05). Our results suggest that repair effects of PRP on the damaged articular cartilage are superior to sodium hyaluronate treatment. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration

Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p < 0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs.     

富血小板血浆调节滑膜炎保护软骨细胞

文题释义：富血小板血浆：是自体生长因子的来源,通过自体全血梯度离心得到。富血小板血浆于1993年Hood等首先提出这一概念,并发现富血小板血浆中含有正常血浆3倍以上的血小板和丰富的生长因子,如血小板衍生生长因子、胰岛素样生长因子、表皮生长因子和血管内皮生长因子等。基质金属蛋白酶：是一类分解细胞外基质组分的锌蛋白酶。它们在有机体生长发育中的细胞外基质逆转与重塑以及疾病中的病理损害起着极为重要的作用。基质金属蛋白酶的表达和活性在不同细胞水平受到严密调控,如细胞因子、生长因子以及激素的调节,基质金属蛋白酶以酶原形式分泌,随后被其他蛋白酶如胞浆素或非蛋白酶类化学物质如有机汞所激活。背景：研究证实,滑膜炎分泌的炎症因子通过加速关节软骨的分解代谢而成为骨性关节炎发展的主要原因,基质金属蛋白酶3和基质金属蛋白酶13在骨性关节炎中起关键作用。目的：探究富血小板血浆治疗骨性关节炎的有效作用机制。方法：分离并提取大鼠膝关节滑膜细胞和软骨细胞分别培养,腹主动脉取血制备富血小板血浆制剂。将滑膜细胞分为空白对照组和大肠杆菌脂多糖处理组,脂多糖处理组用脂多糖刺激滑膜细胞制造滑膜炎模型;再将滑膜炎细胞分为富血小板血浆处理组和未处理组(脂多糖组)。对照组、富血小板血浆处理组和未处理组3组细胞培养24 h后,用ELISA法检测培养基中炎性细胞因子白细胞介素1β、白细胞介素6和肿瘤坏死因子α的水平,另一部分培养基分别处理软骨细胞,培养48 h后,Western blot 检测软骨细胞内Ⅰ型、Ⅱ型胶原蛋白、基质金属蛋白酶3和基质金属蛋白酶13蛋白水平的变化,荧光定量PCR检测基质金属蛋白酶3和基质金属蛋白酶13 mRNA表达水平的变化。结果与结论：①ELISA检测显示,脂多糖处理组滑膜细胞培养基中白细胞介素1β、白细胞介素6和肿瘤坏死因子α的水平显著高于对照组和富血小板血浆处理组(P < 0.01);富血小板血浆处理组培养基中白细胞介素1β、白细胞介素6和肿瘤坏死因子α的水平高于对照组(P < 0.05);②Western blot检测显示,富血小板血浆处理组软骨细胞中Ⅰ型、Ⅱ型胶原蛋白显著高于对照组和脂多糖处理组(P < 0.01),脂多糖处理组低于对照组(P < 0.05);③脂多糖组基质金属蛋白酶3和基质金属蛋白酶13蛋白显著高于富血小板血浆处理组和对照组(P < 0.01);④PCR检测显示脂多糖组基质金属蛋白酶3,13 mRNA显著高于富血小板血浆处理组和对照组;⑤结果表明,富血小板血浆处理能够通过降低滑膜细胞炎性因子白细胞介素1β、白细胞介素6和肿瘤坏死因子α的水平,降低软骨细胞中基质金属蛋白酶3和基质金属蛋白酶13的蛋白和mRNA水平,增加Ⅰ型、Ⅱ型胶原蛋白的表达而发挥保护软骨的作用。ORCID: 0000-0002-0704-4511(陈尉)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

The specific in vitro antibody-mediated retention of bovine serum albumin by porcine hyaline articular cartilage.

Porcine hyaline articular cartilage (HAC) has been used to investigate the interaction of bovine serum albumin (BSA) and anti-BSA with articular collagenous tissues in vitro. There was a marked retention of 125I-labelled BSA by plugs of HAC (a) exposed to rabbit anti-BSA for 1-2 h at 37 degrees C prior to a similar incubation with the antigen, or (b) exposed to the antigen and then to antibody. The specifically retained radiolabelled BSA was localized in or at the articular surface of the plugs. In the absence of specific antibody a relatively small amount of the antigen was retained. Exposure of HAC to multiple cycles of antibody and antigen treatment resulted in an increased retention of the 125I-BSA. There was a concomitant increase in the retention of the anti-BSA and the capacity of the treated plugs to fix complement. The forces that maintained the labelled antigen in the tissue were not readily reversed by excess unlabelled BSA. Pre-formed, soluble BSA/anti-BSA complexes did not appear to penetrate the tissue unless the HAC was first exposed to anti-BSA. The results suggest that the antibody-mediated, surface oriented retention of 125I-BSA results from the formation of immune complexes in the tissue.     

Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-beta1

The study aimed to produce a novel porous gelatin-chondroitin-hyaluronate scaffold in combination with a controlled release of TGF- beta1 and to evaluate its potentials in ectopic cartilage formation. The gelatin-chondroitin-hyaluronate scaffold was developed to mimic the natural extra cellular matrix of cartilage. Gelatin microspheres loaded with TGF- beta1 (MS-TGF beta1) showed a fast cytokine release at initial phase (37.4%) and the ultimate accumulated release was 83.1% by day 18. Then MS-TGF beta1 were incorporated into scaffold. The MSCs seeded on scaffold with or without MS-TGF beta1 were incubated in vitro or implanted subcutaneously in nude mice. In vitro study showed that, compared to the scaffold, the scaffold/MS-TGF beta1 significantly augmented the proliferation of MSCs and GAG synthesis. Three weeks postoperatively histology observation showed that in MSCs/scaffold/MS-TGF beta1 implantation group, cells of newly formed ectopic cartilage were located within typical lacunae and demonstrated morphological characteristics of chondrocytes. Six weeks later the ectopic cartilage grew more and islands of cartilage were observed. The matrix was extensively metachromatic by safranin-O/Fast green staining. Immunohistochemical staining also indicated ectopic cartilage was intensely stained for type II collagen. Instead, in the MSCs/scaffold implantation group, no cartilage-like tissue formed and matrix showed negative or weak positive staining. The percentage of positive staining area was significantly larger in MSCs/scaffold/MS-TGF beta1 group (p<0.05) at each time point. The results indicated that the novel gelatin-chondroitin-hyaluronate scaffold with MS-TGF beta1 could induce the chondral differentiation of MSCs to form cartilage and might serve as a new way to repair cartilage defects.     

The amelioration of cartilage degeneration by ADAMTS-5 inhibitor delivered in a hyaluronic acid hydrogel

Degradation of proteoglycan is the key early event in the development of osteoarthritis (OA). The aggrecanase ADAMTS-5 has been identified as the major enzyme responsible for the degradation and thus is an attractive therapeutic target for OA. However, currently there is no report on using an ADAMTS-5 inhibition strategy for OA treatment. The present study aimed to investigate the synergic effect of combining an ADAMTS-5 inhibitor (114810) with a hyaluronic acid hydrogel (HAX) for OA therapeutics. Two OA models were induced by surgically creating an osteochondral defect or removing the anterior cruciate ligament (ACL) in Sprague–Dawley rats. Human OA cartilage was obtained from total joint replacement patients. Both human and rat OA cartilage showed marked proteoglycan loss with significantly increased ADAMTS-5 expression. The effectiveness of ADAMTS-5 inhibition by 114810 was confirmed by a cartilage explants assay in vitro, which showed that the 114810 halted the aggrecanase-mediated ³⁷⁴ARGS neoepitope released from aggrecan induced by IL-1β stimulation. The in vivo effect of ADAMTS-5 inhibition was assessed by the articular injection of HAX with 114810 into OA knee joints. Evaluated eight weeks after injection, 114810 with HAX significantly promoted the in vivo cartilage healing in the osteochondral defect model, and prevented the progression of degenerative changes in the ACL model. Our results confirmed that ADAMTS-5 is an effective target for OA treatment, and the intra-articular injection of an ADAMTS-5 inhibitor within HAX gel could be a promising strategy for OA treatment.     

Ginsenoside Rb1 enhances peripheral nerve regeneration across wide gaps in silicone rubber chambers

Silicone rubber chambers filled with collagen containing ginsenoside Rb1 (GRb1) were used to repair lesioned rat sciatic nerves with 15-mm gaps between stumps. Six weeks after implantation, histology of the nerve regenerated in the chambers filled with GRb1 and collagen contained larger axons than those in the chambers with collagen only. This study showed that the GRb1 could exert a positive influence on nerve regeneration when using silicone rubber tubes.     

The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects

Partial-thickness cartilage defects, with no subchondral bone injury, do not repair spontaneously, thus there is no clinically effective treatment for these lesions. Although the autologous chondrocyte transplantation (ACT) is one of the promising approaches for cartilage repair, it requires in vitro cell expansion to get sufficient cells, but chondrocytes lose their chondrogenic phenotype during expansion by monolayer culture, leading to de-differentiation. In this study, a heparin-based hydrogel was evaluated and optimized to induce cartilage regeneration with de-differentiated chondrocytes. First, re-differentiation of de-differentiated chondrocytes encapsulated in heparin-based hydrogels was characterized in vitro with various polymer concentrations (from 3 to 20 wt.%). Even under a normal cell culture condition (no growth factors or chondrogenic components), efficient re-differentiation of cells was observed with the optimum at 10 wt.% hydrogel, showing the complete re-differentiation within a week. Efficient re-differentiation and cartilage formation of de-differentiated cell/hydrogel construct were also confirmed in vivo by subcutaneous implantation on the back of nude mice. Finally, excellent cartilage regeneration and good integration with surrounding, similar to natural cartilage, was also observed by delivering de-differentiated chondrocytes using the heparin-based hydrogel in partial-thickness defects of rabbit knees whereas no healing was observed for the control defects. These results demonstrate that the heparin-based hydrogel is very efficient for re-differentiation of expanded chondrocytes and cartilage regeneration without using any exogenous inducing factors, thus it could serve as an injectable cell-carrier and scaffold for cartilage repair. Excellent chondrogenic nature of the heparin-based hydrogel might be associated with the hydrogel characteristic that can secure endogenous growth factors secreted from chondrocytes, which then can promote the chondrogenesis, as suggested by the detection of TGF-β1 in both in vitro and in vivo cell/hydrogel constructs.     

PAR-1 upregulation by trimethyltin and lipopolysaccharide in cultured rat astrocytes

We have previously shown that various protease-activated receptor (PAR) isoforms, mainly PAR-1, are upregulated in reactive astrocytes of rat hippocampus following i.p. administration of trimethyltin (TMT), a neurotoxicant which is known to cause neuronal death and reactive gliosis. In the present paper, we demonstrate that this PAR-1 upregulation was also mimicked in primary cultures of neonatal rat cortex astrocytes after exposure (24 and 48 h) to TMT (10-100 microM). This result suggests that the PAR-1 increase we have observed in vivo may represent a direct effect of TMT on astrocytes rather than a consequence of a complex astrocytic reaction following neuronal death. Furthermore, an evident upregulation of PAR-1 in cultured primary astrocytes also occurred following exposure to lipopolysaccharide (LPS) (a well-known inductor of glial cell activation) whereas other neurotoxic agents (such as staurosporine, hydrogen peroxide and sodium azide), which are known to induce cell death, were unable to determine any PAR-1 variation. Similarly to astrocytes, both TMT and LPS induced an upregulation of PAR-1 in the rat astrocytoma cell line, C6, thus indicating that this phenomenon was independent from microglial cells eventually contaminating astrocyte primary cultures. Furthermore, after exposure to TMT and LPS, the levels of tumor necrosis factor-alpha and interleukin-1beta were also increased in astrocyte cultures, suggesting that the PAR-1 upregulation we have detected may be involved in glial inflammatory response rather than in cell death.     

Platelet-rich plasma induces increased expression of G1 cell cycle regulators, type I collagen, and matrix metalloproteinase-1 in human skin fibroblasts

Platelet-rich plasma (PRP) is derived from fresh whole blood, which contains a high concentration of platelets. Recently, PRP has been used for skin wound healing and rejuvenation. However, the molecular mechanisms underlying PRP-inducing wound healing processes are still largely unknown. The aim of this study is to evaluate the effect of PRP on the expression of G1 cell cycle regulatory proteins, type I collagen, matrix metalloproteinase-1 (MMP-1), and MMP-2 in human skin fibroblasts (HSF). We performed a cell proliferation and a migration assay, immunoblotting, and a chloramphenicol acetyltransferase (CAT) assay in PRP-treated human skin fibroblasts. PRP treatment induced increased rates of cell proliferation and cell migration. Expression of cyclin A protein was increased by a low concentration (0.5%) of PRP-treated HSF. In addition, expression of Rb, cyclin E, and cyclin-dependent kinase 4 proteins was increased by a high concentration (5%) of PRP-treated HSF. High concentration of PRP induced an up-regulation of type I collagen, MMP-1, and MMP-2 expression in HSF. Taken together, PRP treatment induced an increase in expression of G1 cell cycle regulators, type I collagen and MMP-1, thereby accelerating the wound healing process.     

Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor

Diabetic skin ulcer is difficult to heal due to the lack of cellular and molecular signals required for normal wound repair. Emulsion electrospinning was adopted to imbed basic fibroblast growth factor (bFGF) into ultrafine fibers with a core-sheath structure to promote the wound healing process. An initially burst release as low as 14.0 ± 2.2% was achieved, followed by gradual release for around 4 wk. In vitro investigations on mouse embryo fibroblasts indicated that bFGF-loaded fibrous mats enhanced cell adhesion, proliferation, and secretion of extracellular matrix (ECM). Skin wounds were created in the dorsal area of diabetic rats for in vivo evaluation of skin regeneration after covered with bFGF-loaded fibrous mats. Compared with fibrous mats infiltrated with free bFGF, bFGF-loaded scaffolds revealed significantly higher wound recovery rate with complete re-epithelialization and regeneration of skin appendages. Higher density and mature capillary vessels were generated during 2 wk after treatment with bFGF-loaded fibers, and there was no fiber fragment observed in the histological sections at week 4 after operation. The gradual release of bFGF from fibrous mats enhanced collagen deposition and ECM remodeling, and the arrangement and component of collagen fibers were similar to normal tissues. The above results demonstrate the potential use of bFGF-loaded electrospun fibrous mats to rapidly restore the structural and functional properties of wounded skin for patients with diabetic mellitus.     

FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

A prolonged period of T‐cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T‐cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cell‐penetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T‐cell regeneration in patients following HSCT.     

Skull bone regeneration in nonhuman primates by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel

The objective of this study was to investigate the feasibility of biodegradable gelatin hydrogels as the controlled-release carrier of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration at a skull defect of nonhuman primates. Hydrogels with 3 different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. A critical-sized defect (6 mm in diameter) was prepared at the skull bone of skeletally mature cynomolgus monkeys, and gelatin hydrogels incorporating various doses of BMP-2 were applied to the defects. When the bone regeneration was evaluated by soft radiography and bone mineral density (BMD) examinations, the gelatin hydrogel incorporating BMP-2 exhibited significantly higher osteoinduction activity than did an insoluble bone matrix that incorporated BMP-2 (one of the best osteoinduction systems), although the activity depended on the water content of hydrogels. BMD enhancement was highest for the gelatin hydrogel that had a water content of 97.8 wt% among all types of hydrogels. Moreover, the gelatin hydrogel enabled BMP-2 to induce the bone regeneration in nonhuman primates even at low doses. We conclude that the controlled release of BMP-2 for a certain time period was essential to inducing the osteoinductive potential of BMP-2.     

Cytoplasmic p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal cord injury in rats

p21(Cip1/WAF1), known as a cell-cycle inhibitory protein, facilitates neurite outgrowth from neurons when present in the cytoplasm. The molecular mechanism of this action is that p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity. As myelin-derived inhibitors of axonal outgrowth act on neurons by activating Rho, that is responsible for the lack of spontaneous regeneration of the injured central nervous system (CNS), Rho-kinase may be a good molecular target against injuries in the CNS. In this study, we delivered TAT-fusion protein of cytoplasmic p21(Cip1/WAF1) locally after dorsal hemisection of the thoracic spinal cord in rats. The treatment significantly stimulated axonal regeneration and recovery of hindlimb function, and inhibited the cavity formation in the spinal cord after the injury. Cytoplasmic p21(Cip1/WAF1) may provide a potential therapeutic agent that produces functional regeneration following CNS injuries.     

Regulation of cannabinoid CB1 receptors in the central nervous system by chronic cannabinoids

Marijuana produces a number of characteristic behaviors in humans and animals, including memory impairment, antinociception, and locomotor and psychoactive effects. However, tolerance and dependence to cannabinoids develops after chronic use, as demonstrated both clinically and in animal models. The potential therapeutic benefits of certain cannabinoid-mediated effects, as well as the use of marijuana for its psychoactive properties, has raised interest in understanding the cellular adaptations produced by chronic administration of this class of drugs. The primary active constituent of marijuana, delta9-tetrahydrohydrocannabinol (THC), binds to specific G-protein-coupled receptors. The central nervous system (CNS) effects of THC are mediated by CB1 receptors, which couple primarily to inhibitory G-proteins. High levels of CB1 receptors are found in the basal ganglia, hippocampus, cortex, and cerebellum, consistent with the profile of behavioral effects. Studies over the past decade have determined that CB1 receptors undergo downregulation and desensitization following chronic administration of THC or synthetic cannabinoid agonists. In general, these adaptations are regionally widespread and of considerable magnitude, and are thought to contribute to tolerance to cannabinoid-mediated behavioral effects. Adaptation at the effector level has been more difficult to characterize, although it appears that alterations in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity may be particularly important in cannabinoid dependence. A striking characteristic of CB 1 receptor adaptation is the region dependence of the magnitude and rate of development of downregulation and desensitization. These regional differences may provide interesting insights into the mechanisms of CB1 receptors receptor signaling in different brain regions. Moreover, region-specific adaptations in CB1 receptors following chronic cannabinoid administration may produce differential adaptations at the in vivo level.