The enhanced osteogenesis and osteointegration of 3-DP PCL scaffolds via structural and functional optimization using collagen networks

Optimal balance between biological activity and mechanical stability should be meticulously considered during scaffold design for bone tissue engineering applications. To fabricate an individualized construct with biomechanical and biological functionality for bone tissue regeneration, a polycaprolactone–collagen (PCL–COL) composite construct was developed through the combination of three-dimensional printing (3-DP) technology and biomimetic collagen matrix incorporation, with a 3-DP PCL framework maintaining the mechanical stability and a porous collagen matrix improving the biological activity. The results indicate that the compressive modulus of the composite constructs increased synergistically (over 40 MPa), providing sufficient mechanical support during new bone formation. On the other hand, the collagen matrix with a micro-porous architecture structurally increased scaffold areas and provided cellular adhesion sites, allowing for the functional construction of a favorable 3D microenvironment for BMSC adhesion, proliferation and extracellular matrix production. Moreover, critical-sized long bone defect (CSD) implantation demonstrated that the optimized composite constructs could promote bone tissue regeneration (5.5-fold) and bone-material osteointegration (4.7-fold), and decrease fibrosis encapsulation, compared to pristine PCL. The results indicate that these biomimetically ornamented PCL–COL constructs exhibit favorable mechanical properties and biological functionality, demonstrating great potential as an effective bone graft substitute for bone defect treatment. Meanwhile, they can also harness the advantages of 3-DP technology and a collagen-based functionalized strategy, facilitating the creation of customized and functional PCL–COL constructs for clinical translation.

Optimal balance between biological activity and mechanical stability should be meticulously considered during scaffold design for bone tissue engineering applications.     

组织工程膀胱支架材料：应用现状及血管化策略

背景：随着组织工程膀胱研究的日益改进,组织工程膀胱组织在植入体内后的血管化问题,引起了构建组织工程膀胱者的极大关注。目的：结合近年来的相关文献,就组织工程膀胱支架材料的选择、设计与应用,支架材料植入体内后的血管化问题作一综述。方法：由第一作者应用计算机检索PubMed数据库2000年1月至2014年9月的相关文章,检索词为“tissue engineering,bladder,biomaterials/scaffolds,vascularization”;同时检索中国期刊数据库2000年1月至2014年9月的相关文章,检索词为“组织工程,膀胱,支架材料,血管化”,选择文章内容与生物支架材料在组织工程膀胱中的应用及组织工程膀胱血管化相关。结果与结论：组织工程膀胱中目前采用的支架材料主要包括天然生物材料和人工合成聚合物两大类。当前膀胱组织工程研究最主要的目标仍然是：制备接种细胞的最佳支架,确定干细胞的最佳来源,探索干细胞最优分化方式和促进植入支架新生血管和神经的再生,其中促进支架材料的血管化和构建复杂的组织是最具有挑战性的。目前来说,使支架上附着的内膜细胞精确的定向增殖、迁移和分化仍然很难控制。尽管血管网对于细胞和组织的营养供应和代谢废物清除是必要的,但促进血管生成或血管发生的策略仍有限。     

神经导管生物材料在神经修复中的应用

背景：神经导管是由天然或人工合成材料制成的、用于桥接神经断端的组织工程支架材料,具有引导和促进神经再生作用。目的：总结近年来常用的神经导管生物材料在神经修复中的应用。方法：由作者应用计算机检索维普数据库中与神经导管生物材料在神经修复中应用有关的文章,检索时限2002-01/2010-12。检索关键词：神经导管;生物材料;神经损伤;神经修复;神经再生。纳入标准：与神经导管生物材料在神经修复中应用有关的文章。排除标准：重复研究或较陈旧文献。根据纳入排除标准共保留相关文献30篇。结果与结论：非生物降解材料由于其不可吸收性和对再生神经的远期不良影响使临床应用受到限制。生物降解材料在神经再生完成后可在体内降解吸收,无需二次手术取出,但目前未能利用生物降解材料完全仿制出具有天然神经结构的支架。生物衍生材料生物相容性好、排异反应小,可提供细胞外基质、胶原,起支架作用,但缺血后存在管形塌陷、再生不良、吸收瘢痕组织、增生及粘连等问题。神经导管生物材料在神经修复中的应用前景广阔,但单用一类材料难以制作出理想的神经导管生物材料,通过结合各类材料的优点,与神经营养因子、细胞外基质成分和许旺细胞等联合应用,制备新型具有生物活性的导管材料,将有利于神经修复进一步发展。     

Preparation of differently sized injectable collagen micro-scaffolds

Collagen scaffolds have been widely used as biomaterials for tissue engineering. In general, application of scaffolds requires surgery. In this study, we describe a new and simple technique to prepare porous micro-scaffolds from type I collagen fibrils which can be injected, thus preventing surgery. The size of the micro-scaffolds could be easily controlled using sieves with varying cut-offs. EDC-NHS crosslinking was essential to stabilize the collagen micro-scaffolds. Micro-scaffolds were highly porous and could be injected through small diameter needles (18-21 gauge). Collagen micro-scaffolds may be used as injectables for the local delivery of effector molecules and/or cells, thus creating specific niches to enhance local tissue regeneration.     

骨髓间充质干细胞在膀胱脱细胞基质上的生长及分化

背景:传统方法多采用平滑肌细胞和移行上皮细胞构建组织工程膀胱,并进行支架材料的双面种植,但由于平滑肌细胞取材培养困难,且体外传代有限,双面种植较为困难.目的:实验拟验证以骨髓间充质干细胞及膀胱脱细胞基质构建组织工程膀胱的可行性.设计:基础实验研究.单位:中山大学附属第二医院林百欣医学研究中心.材料:实验于2006-03/2007-05在中山大学附属第二医院林百欣医学研究中心完成.实验室级别为卫生部部属医院开放实验室.1月龄SD大鼠,雌雄不限,体质量80～100g,由中山大学实验动物中心提供.新鲜猪膀胱取自南方医科大学动物实验中心.方法:采用全骨髓培养连续贴壁法体外培养大鼠骨髓间充质干细胞,并应用流式细胞仪检测其表面抗原.采用去污剂洗涤法制备猪膀胱脱细胞基质,并测定其纯度及特性.将第3代骨髓间充质干细胞接种到膀胱脱细胞基质上,以添加25 ng/L血管内皮生长因子(VEGF165)的培养液进行体外、体内复合培养,检测其相容性.体内实验以细胞单独培养为对照,体外实验以未植入细胞的材料为对照,并模拟合适的微环境诱导骨髓间充质干细胞生长分化,分别在4,8周后取出动物体内的复合材料行组织切片检查,并进行免疫组织化学角蛋白染色,检测上皮细胞再生情况.主要观察指标:骨髓间充质干细胞与膀胱脱细胞基质的生物相容性.结果:①采用全骨髓法成功培养出骨髓间充质干细胞,行流式细胞仪检测细胞表面抗原显示,传代第3代细胞CD29阳性细胞为99.43%.②制备的膀胱脱细胞基质具有良好的生物特性,镜下见均质状态的基质和细丝状的胶原纤维.体内外相容性实验表明骨髓间充质干细胞与膀胱脱细胞基质具有良好的相容性,细胞生长状态良好.③4周后组织切片检查可见组织中较多炎症细胞浸润,胶原及弹性纤维排列紧密,免疫组织化学角蛋白染色见有薄层、不连续的单层上皮生长,8周后可见组织中已无明显炎症细胞浸润反应,胶原及弹性纤维排列紧密,免疫组织化学角蛋白染色见有薄层、连续的多层上皮生长.结论:骨髓间充质干细胞与膀胱脱细胞基质具有良好的生物相容性,并且在体内与周围组织有良好的生物相容性,可以作为构建组织工程膀胱的材料.     

神经导管生物材料在神经修复中的应用

背景:神经导管是由天然或人工合成材料制成的、用于桥接神经断端的组织工程支架材料,具有引导和促进神经再生作用.目的:总结近年来常用的神经导管生物材料在神经修复中的应用.方法:由作者应用计算机检索维普数据库中与神经导管生物材料在神经修复中应用有关的文章,检索时限2002-01/ 2010-12.检索关键词:神经导管;生物材料;神经损伤;神经修复;神经再生.纳入标准:与神经导管生物材料在神经修复中应用有关的文章.排除标准:重复研究或较陈旧文献.根据纳入排除标准共保留相关文献30篇.结果与结论:非生物降解材料由于其不可吸收性和对再生神经的远期不良影响使临床应用受到限制.生物降解材料在神经再生完成后可在体内降解吸收,无需二次手术取出,但目前未能利用生物降解材料完全仿制出具有天然神经结构的支架.生物衍生材料生物相容性好、排异反应小,可提供细胞外基质、胶原,起支架作用,但缺血后存在管形塌陷、再生不良、吸收瘢痕组织、增生及粘连等问题.神经导管生物材料在神经修复中的应用前景广阔,但单用一类材料难以制作出理想的神经导管生物材料,通过结合各类材料的优点,与神经营养因子、细胞外基质成分和许旺细胞等联合应用,制备新型具有生物活性的导管材料,将有利于神经修复进一步发展.     

Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin–mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.OCIS codes: (190.1900) Diagnostic applications of nonlinear optics, (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy     

Airway tissue engineering: an update

Introduction: Prosthetic materials, autologous tissues, cryopreserved homografts and allogeneic tissues have thus far proven unsuccessful in providing long-term functional solutions to extensive upper airway disease and damage. Research is therefore focusing on the rapidly expanding fields of regenerative medicine and tissue engineering in order to provide stem cell-based constructs for airway reconstruction, substitution and/or regeneration.

Areas covered: Advances in stem cell technology, biomaterials and growth factor interactions have been instrumental in guiding optimization of tissue-engineered airways, leading to several first-in-man studies investigating stem cell-based tissue-engineered tracheal transplants in patients. Here, we summarize current progress, outstanding research questions, as well as future directions within the field.

Expert opinion: The complex immune interaction between the transplant and host in vivo is only beginning to be untangled. Recent progress in our understanding of stem cell biology, decellularization techniques, biomaterials and transplantation immunobiology offers the prospect of transplanting airways without the need for lifelong immunosuppression. In addition, progress in airway revascularization, reinnervation and ever-increasingly sophisticated bioreactor design is opening up new avenues for the construction of a tissue-engineered larynx. Finally, 3D printing is a novel technique with the potential to render microscopic control over how cells are incorporated and grown onto the tissue-engineered airway.     

功能性组织工程皮肤附属物再生对排汗和新陈代谢的意义

背景：排汗可将一些溶解于汗液的代谢产物带出体外,在排泄中起辅助作用。现代医学目前还无法恢复严重创伤或烧伤患者的汗腺,如何解决皮肤汗腺的再生及如何培养具有皮肤附属物的“功能性组织工程皮肤”问题已成为当今医学研究中的热点。目的：探讨机体的新陈代谢、汗液的代谢方式,分析组织工程皮肤功能性附属物汗腺再生研究的现状。方法：收集组织工程皮肤功能性附属物再生,特别是排汗的相关研究,分析机体的新陈代谢与排泄方式,汗液代谢的物质和方式,以及目前组织工程皮肤附属物汗腺再生研究的现状。结果与结论：汗液作为排泄系统中重要的一员在维持机体内部的动态平衡,防止代谢产物对身体的损害方面具有不可忽视的地位。理想的功能性组织工程皮肤应具有分泌汗液、排泄废物、调节体温和维持皮肤动态平衡的作用,在大面积烧伤后皮肤创伤修复和重建过程中起着至关重要的作用。已有研究将干细胞与汗腺细胞进行共培养,然后移植到瘢痕创面上进行重建汗腺组织,取得了一定的成功。     

Full‐thickness oesophageal regeneration in pig using a polyurethane mucosal cell seeded graft

Malignant oesophageal pathology typically requires resection of a portion of oesophagus. The aim of this study was to investigate attachment and growth of swine oesophageal mucosal cells on electrospun synthetic nanofibre matrices of varying chemistries and to determine whether a mucosal‐seeded graft, in a swine animal model, could induce regeneration. Swine mucosal oesophageal cells were isolated and seeded them onto five different matrix materials. Matrix samples were cultured for up to 14 days, after which matrices were analysed for cell attachment. Attachment varied for each of the matrix materials tested, with the most rigid showing the lowest levels of attachment. Importantly, sections of these matrices illustrated that multiple layers of mucosal cells formed, mimicking endogenous oesophageal structure. A tdTomato reporter line (mucosal^tdt cells) was created to enable cell tracking. As polyurethane matrix was found optimal through in vitro testing, a graft was prepared using mucosal^tdt cells, along with an unseeded control, and implanted into swine for determination of oesophageal regeneration. Mucosal seeded polyurethane grafts initiated full thickness regeneration of the oesophagus, including epithelial, submucosal, and skeletal muscle layers which were highly vascularized. Interestingly, an unseeded graft showed similar regeneration, indicating that the role of cells in the process of oesophageal regeneration is still unclear. The electrospun polyurethane matrix does appear suitable for multilayered cellular attachment and growth of oesophageal mucosal cells, and implantation of polyurethane grafts initiated full thickness regeneration of the oesophagus, indicating potential for oesophageal reconstruction in humans. Copyright © 2016 John Wiley & Sons, Ltd.     

Osteoinductive biomaterials--properties and relevance in bone repair

The need for bone tissue regeneration is continuously expanding due to the improvement of life quality and the consequent increase in life expectancy. Although natural bone grafts have shown excellent clinical successes, their use is associated with some important drawbacks, limited availability being one of the most important. Cell- and growth-factor based tissue engineering provides a promising alternative to natural bone grafts; however, the performance of tissue-engineered constructs often depends on the used carrier. An important challenge in the field of bone regeneration is the development of synthetic bone graft substitutes that are "intelligent" in that they are able to instruct the in vivo environment to form bone. A group of potentially "intelligent" bone graft substitutes are osteoinductive biomaterials. In this paper, background on the phenomenon of osteoinduction and an overview of synthetic biomaterials with osteoinductive potential are given. Furthermore, we elaborate on physicochemical properties of biomaterials that are of influence on their osteoinductive potential. Finally, we discuss the relevance of osteoinductivity of biomaterials in the repair of clinically relevant bone defects.     

不同材料构建组织工程软骨及支架的应用

背景：组织工程技术的发展为软骨的再生和修复提供了新的途径,根据软骨自身的结构和特点,作为人工软骨的替代材料和支架材料应具有良好的生物力学性能。目的：总结运动性关节软骨损伤修复材料及其支架材料的应用进展及其生物替代材料的生物力学特征,评价目前组织工程软骨材料应用的性能及发展前景。方法：以＂组织工程;软骨组织;支架材料;生物相容性＂为关键词,应用计算机检索维普数据库和PubMed数据库中1990-01/2011-04关于组织工程软骨应用研究的文章,纳入与有关生物材料与组织工程软骨相关的文章;排除重复研究或Meta分析类文章。以24篇文献为主重点进行了讨论组织工程软骨材料的种类、性能及其应用效果和前景。结果与结论：目前关节软骨修复领域以自体软骨移植效果为最佳,骨髓基质干细胞在离体试验及动物实验中研究较多,在临床应用中较少,尚在探索阶段。支架材料的应用比较繁复,天然材料、人工合成材料以及复合材料都存在一定的不足,虽然复合材料成为研究的热点,但是某些性能并不能很好地符合支架要求,并且在机体内这些材料所带来的长期影响还不能预见,这就迫切需要新材料的出现,来更好地满足组织软骨织支架的要求,达到修复和重建的目的。     

碳纳米管增强型天然复合材料神经导管修复大鼠副神经缺损

背景:近年来,纳米技术在组织重建领域的应用研究十分活跃.碳纳米材料应用于骨组织修复的相关报道较多,但应用于周围神经系统修复的报道较少.目的:应用功能性修饰的碳纳米管作为增强成分改善几丁糖/胶原复合材料神经导管的理化和生物性能,探讨以功能性修饰的碳纳米管增强型复合材料神经导管修复周围神经缺损的疗效.设计、时间及地点:同体自身对照动物实验,于2005-02/2006-11在上海交通大学医学院El腔组织工程实验室(上海市口腔重点实验室)和上海交通大学薄膜与微细技术实验室(教育部重点实验室)完成.材料:将碳纳米管与2%的酸溶性几丁糖溶液及胶原按一定比例充分混合,涂覆在模具上,经红外加热成型并达到预期厚度后,干燥脱模制备碳纳米管增强型复合材料导管.以不含碳纳米管的几丁糖,胶原复合材料导管为对照材料.方法:成年健康雄性Sprague-Dawley大鼠80只,制备4 mm副神经缺损,实验材料组和对照材料组分别应用相应神经导管进行修复.自体神经移植组将切除的神经原位吻接于断端,空白对照组则仅将神经切除掉2 mm,不做处理.左侧为实验侧,右侧为正常对照侧.主要观察指标:通过神经电生理学、肌肉功能及组织学检测手段对其修复效果进行评价.结果:应用碳纳米管增强型复合材料神经导管可有效地重建副神经缺损大鼠斜方肌的运动功能.术后再生神经电生理与组织学指标检测结果与白体神经移植的疗效相当,部分指标结果超过自体神经移植.结论:碳纳米管增强型复合材料神经导管是桥接修复周围神经的理想材料.     

In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy

We implemented in situ time-lapse Second Harmonic Generation (SHG) microscopy to monitor the three-dimensional (3D) self-assembly of collagen in solution. As a proof of concept, we tuned the kinetics of fibril formation by varying the pH and measured the subsequent exponential increase of fibril volume density in SHG images. We obtained significantly different time constants at pH = 6.5 ± 0.3 and at pH = 7.5 ± 0.3. Moreover, we showed that we could focus on the growth of a single isolated collagen fibril because SHG microscopy is sensitive to well-organized fibrils with diameter below the optical resolution. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials.     

骨髓间充质干细胞-聚乙醇酸支架复合体种植动物皮下构建组织工程化小口径血管(英文)

背景:课题组的前期工作已证实骨髓间充质干细胞可以诱导分化为血管内皮细胞和血管平滑肌细胞,并证实所诱导的细胞和胶原包埋的聚乙醇酸支架具有良好的组织相容性。目的:探讨利用动物皮下作为生物反应器构建小口径组织工程化血管的可行性。方法:骨髓间充质干细胞诱导分化为血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸支架表面,然后将细胞-支架复合体种植于动物皮下,构建小口径组织工程化血管。结果与结论:人工血管组织学观察见管壁结构清晰,其大体结构和天然血管相似,可承受26.6kPa的血管腔内压力不破裂。皮下培养8周免疫荧光观察Brdu标记的部分细胞核呈现明亮的黄绿色荧光。结果说明利用动物的皮下作为生物反应器,采用静态培养的方式构建小口径组织工程化血管是可行的。     

聚乙醇酸-乳酸共聚物复合Ⅱ型胶原和生长因子体内构建组织工程软骨

背景:软骨组织工程的发展为处理关节软骨损伤提供了新的思路和方法,使体内构建组织工程软骨得以实现.目的:观察骨髓基质干细胞种植到复合胶原和生长因子的聚乙醇酸-乳酸共聚物(poly-lactide-co-glycolic acid,PLGA)生物材料,再种植到大鼠体内构建组织工程软骨组织的可行性.方法:相分离法制作PLGA,复合Ⅱ型胶原和碱性成纤维细胞生长因子,转化生长因子β1.将第3代的骨髓基质干细胞种植到复合材料上.36只SD大鼠随机分为实验组、对照组、空白组,分别于肌袋内植入骨髓基质干细胞/复合生长因子和胶原的PLGA、复合生长因子和胶原的PLGA、复合胶原的PLGA,于术后第4,8,12周取材观察细胞的定向分化及生长情况,包括大体观察、苏木精-伊红染色、甲苯胺蓝染色、Ⅱ型胶原染色、扫描电镜观察.结果与结论:大体观察可见实验组材料有类软骨样组织生长,而对照组和空白组则仅见纤维组织生长.各种染色及电镜观察显示:实验组复合物内可见多的成软骨细胞及少量的破骨细胞.实验组甲苯胺蓝染色和Ⅱ型胶原染色为阳性,对照组和空白组均为阴性.从而证明胶原修饰的PLGA生物材料具有较好的细胞相容性;骨髓基质干细胞种植到复合胶原和生长因子的PLGA生物材料上在大鼠体内可构建组织工程软骨复合组织.     

Collagen engineering for biomaterial use

Collagen is a typical biological macromolecule having been utilized for a long period of time as a material like cellulose. However, its application is becoming comprehensive, ranging from classical applications such as the leather, gelatine and food industries to the current one, namely, biomaterial and biotechnological uses. The diversification of collagen applications was enhanced by two factors, the accumulation of the scientific knowledge that permitted proper engineering of collagen for a biomaterial use, and the demand for new biomaterials with characteristic biological properties including interaction with cells. The collagen engineering described in this paper is designed for biomaterial use, based on the fundamental chemical and biological properties of collagen; however, it would be useful also for other applications apart from biomaterials.     

间充质干细胞构建组织工程化气管的研究与应用

背景：间充质干细胞诸多特有的良好生物学特性使得其在组织工程化气管研究中成为理想的种子细胞来源。目的：总结间充质干细胞在构建组织工程化气管领域的研究现状、进展、存在的问题及展望。方法：由作者检索PubMed数据库及CNKI数据库1979至2012年,在英文标题和摘要中以＂mesenchymal stem cells,tissue-engineered trachea＂和＂tracheal chondrocytes,tracheal epithelial cells,tracheal vascular endothelial cells＂检索,中文文献检索中以＂间充质干细胞、组织工程化气管、气管软骨细胞、气管上皮细胞和气管血管内皮细胞＂为关键词,选择与组织工程化气管相关的文章,同一领域文献则选择近期发表或发表在权威杂志的文章,共纳入51篇。结果与结论：组织工程化气管种子细胞研究主要包括软骨细胞、上皮细胞和血管内皮细胞。实验已证实,间充质干细胞可定向分化为软骨细胞,细胞因子在诱导间充质干细胞分化为软骨过程中起着关键作用。目前尚未发现合适的诱导因子能特异性诱导间充质干细胞定向分化气管上皮细胞。有研究发现间充质干细胞具有向上皮细胞分化的潜能。间充质干细胞在体内或体外特殊条件下可诱导分化为血管内皮细胞,为间充质干细胞作为种子细胞分化血管内皮细胞应用于组织工程化气管起到借鉴作用。     

胶原/透明质酸膜与明胶海绵支架材料力学及组织相容性的比较

背景:课题组前期实验证明胶原/透明质酸膜具有良好的力学性能和组织相容性.目的:观察复合材料胶原/透明质酸膜及明胶海绵的生物学性能.方法:应用材料复合交联的实验方法构建胶原/透明质酸膜并测定胶原/透明质酸膜、明胶海绵支架的力学性能.将支架材料种植于兔皮下,按照2,4,6,8,12 周不同时间点评价材料在体内的降解情况和组织相容性.结果与结论:①成功制备了胶原/透明质酸膜.②胶原/透明质酸膜具有较好的韧性和抗张强度,明胶海绵的力学性能不够理想.③两种材料在体内的降解均符合生物材料的组织反应过程,胶原/透明质酸膜在体内12 周可完全降解,明胶海绵约6 周完全降解.④胶原/透明质酸膜与平滑肌细胞的黏附率高,细胞的增殖和代谢状况较好,而明胶海绵的细胞黏黏附和增殖率相对较低.说明胶原/透明质酸膜具有较好的生物学性能.     

Dehydrothermal treatment of collagen influences on bone regeneration by octacalcium phosphate (OCP) collagen composites

We have engineered a scaffold constructed of synthetic octacalcium phosphate (OCP) and collagen composites (OCP-collagen) and report that OCP-collagen significantly enhanced bone regeneration more than the implantation of OCP. We hypothesized that the dehydrothermal treatment (DHT) during the fabrication of OCP-collagen might influence bone regeneration by OCP-collagen. To examine this hypothesis, bone regeneration by the implantation of OCP-collagen with DHT [OCP/Col(+)] was compared with that by OCP-collagen without DHT [OCP/Col(-)]. It was confirmed that both OCP/Col(+) and OCP/Col(-) contained the characteristics of OCP structure in X-ray diffraction. Before implantation, calcium deposition derived from OCP was observed within the collagen of both OCP/Col(+) and OCP/Col(-) by undecalcified histological sections. OCP/Col(+) or OCP/Col(-) was implanted into the critical-sized defects in rat crania. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. N-Bone% treated with OCP/Col(+) was significantly higher than that with OCP/Col(-) at 4 and 12 weeks after implantation, because fast degradation of the implanted collagen of OCP/Col(-) elicited disappearance of the scaffold for bone regeneration. The stiffness of the calcified collagen in OCP-collagen would be more important than the existence of calcified collagen to enhance the bone regeneration by OCP-collagen composites. The present study suggests that the dehydrothermal treatment would influence effective bone regeneration by OCP-collagen.