Establishment and effects of allograft and synthetic bone graft substitute treatment of a critical size metaphyseal bone defect model in the sheep femur

Assessment of bone graft material efficacy is difficult in humans, since invasive methods like staged CT scans or biopsies are ethically unjustifiable. Therefore, we developed a novel large animal model for the verification of a potential transformation of synthetic bone graft substitutes into vital bone. The model combines multiple imaging methods with corresponding histology in standardized critical sized cancellous bone defect. Cylindrical bone voids (10 ml) were created in the medial femoral condyles of both hind legs (first surgery at right hind leg, second surgery 3 months later at left hind leg) in three merino‐wool sheep and either (i) left empty, filled with (ii) cancellous allograft bone or (iii) a synthetic, gentamicin eluting bone graft substitute. All samples were analysed with radiographs, MRI, μCT, DEXA and histology after sacrifice at 6 months. Unfilled defects only showed ingrowth of fibrous tissue, whereas good integration of the cancellous graft was seen in the allograft group. The bone graft substitute showed centripetal biodegradation and new trabecular bone formation in the periphery of the void as early as 3 months. μCT gave excellent insight into the structural changes within the defects, particularly progressive allograft incorporation and the bone graft substitute biodegradation process. MRI completed the picture by clearly visualizing soft tissue ingrowth into unfilled bone voids and presence of fluid collections. Histology was essential for verification of trabecular bone and osteoid formation. Conventional radiographs and DEXA could not differentiate details of the ongoing transformation process. This model appears well suited for detailed in vivo and ex vivo evaluation of bone graft substitute behaviour within large bone defects.     

兔股骨髁临界性骨缺损动物模型制备及临界骨缺损值

文题释义：临界性骨缺损：首先定义为自然状况下骨缺损不进行任何处理无法自愈的最短的骨缺损尺寸。随后考虑到观察实验动物完整的生命周期是非常困难的,将临界性骨缺损值定义为在实验期间物种不能自行愈合的最短骨缺损尺寸。 动物模型：是在医学研究中建立的模拟人类疾病表现的动物,骨组织工程中建立临床相关的测试动物模型来研究材料的生物相容性、降解、力学性能以及与宿主组织的相互作用,是体外实验和人体临床试验之间的关键一步。 背景：兔股骨远端骨缺损模型被研究者们广泛用于骨缺损替代骨组织工程材料的测试,但对于兔股骨髁圆柱形骨缺损模型的大小文献报道不一,直径分布在5-9 mm,深度8-12 mm,目前尚无统一的标准。 目的：建立兔股骨髁不同尺寸骨缺损模型,确定兔股骨髁临界性骨缺损尺寸。 方法：6月龄雄性新西兰白兔18只,随机分为3组,每组各6只,分别建立骨缺损模型,骨缺损直径依次为5,6,7 mm,深度均为10 mm,双侧手术,共计12侧。分别于术后第1天及术后第4,8,12周行CT扫描及三维重建,CT-Hedberg评分评价骨缺损愈合情况;于术后12周处死新西兰白兔,取出股骨髁缺损样本,通过大体观察和苏木精-伊红染色分析缺损区愈合情况。实验方案经徐州医科大学实验动物道德伦理委员会批准。 结果与结论：①术后所有兔均存活,术后12周大体观察示：直径5 mm组缺损由新生骨组织充填,股骨髁塑形良好,骨缺损基本完全修复;直径6 mm组、直径7 mm组骨缺损区可见明显凹陷,新生骨组织较少,骨缺损未修复;②CT图像示：术后第4,8周,直径5 mm组缺损区逐渐减小,断端桥接;直径6 mm、直径7 mm组缺损区仅周边有少量新生骨长入,缺损面积较前稍减小;术后第12周可见直径5 mm组皮质骨结构完整、连续,骨缺损基本完全修复;直径6 mm组骨缺损部分修复;直径7 mm组缺损未修复,仍可见明显缺损空腔存在;③CT-Hedberg评分显示,术后各时间点直径6 mm组评分显著低于直径5 mm组(P < 0.05);与直径7 mm组比较差异无显著性意义(P > 0.05);④组织学结果示：术后12周直径5 mm组缺损区出现排列不规则的骨小梁结构,并可见大量新生骨组织填充,其他2组在骨缺损周边可见部分新生骨小梁存在,但缺损区新生骨组织填充较少;⑤结果说明,在12周的实验观察期内,在缺损深度同为10 mm的条件下,直径>6 mm的股骨髁缺损未能自行愈合,而直径<6 mm的股骨髁缺损基本完全修复。此结果符合临界骨缺损的标准,故直径6 mm可作为兔股骨髁临界骨缺损值。 ORCID: 0000-0002-1257-965X(徐石庄) 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     

Validation of a femoral critical size defect model for orthotopic evaluation of bone healing: a biomechanical, veterinary and trauma surgical perspective

Numerous in vivo studies have been conducted to investigate bone regeneration in orthotopic defect models, but a reliably standardized critical-size defect (CSD) model in small animals is still lacking in tissue-engineering research. Utilizing the expertise of trauma surgeons, veterinary surgeons, and engineers, we evaluated the optimal fixation strategy for in vivo application in terms of surgical suitability and conducted biomechanical studies for 3 fixation devices. Fixation strategies were an external fixation device made of polymethylmethacrylate, widely used in animal care; a self-constructed external clamp-fixation device, designed and manufactured using rapid prototyping techniques; and commercially available 1.2-mm titanium plates used in hand surgery. The CSD was 6 mm in size. Biomechanical testing included compression, 4-point bending, and torsion tests. The surgical procedure was optimized in vitro and validated in a clinical setting in athymic rats in vivo. Despite differences in the results of the biomechanical tests, all fixation devices tested proved suitable for the intended purpose. In conclusion, the evaluated model for stabilizing a CSD in a rat's femur can reliably be used for standardized bone regeneration studies in small animals.     

In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model

Calcium sulfate as a bone graft substitute is rapidly resorbed in vivo releasing calcium ions but fails to provide long-term three-dimensional framework to support osteoconduction. The setting properties of calcium sulfate however allow it to be applied in a slurry form making it easier to handle and apply in different situations. This study examines the in vivo response of calcium sulfate alone and as a carrier for a coralline hydroxyapatite in an established bilateral corticocancellous defect model in rabbits. Defects were filled flush to the anterior cortex with a resorbable porous ceramic alone and in combination with calcium sulfate slurry, calcium sulfate slurry alone or calcium sulfate pellets and examined at time points up to 52 weeks. Specimens where assessed using Faxitron X-ray, light and electron microscopy. Calcium sulfate in either slurry or pellet form does indeed support new bone formation alone however, complete filling of the bone defect is not observed. Calcium sulfate in slurry form does however improve the surgical handling of particulate bone graft substitutes such as Pro Osteon 200 R, which remained as an osteoconductive scaffold for up to 52 weeks and may have played an important role in the ultimate closure of the cortical windows.     

Histological evaluation of an impacted bone graft substitute composed of a combination of mineralized and demineralized allograft in a sheep vertebral bone defect

Demineralized bone matrix (DBMs) preparations are a potential alternative or supplement to autogenous bone graft, but many DBMs have not been adequately tested in clinically relevant animal models. The aim of current study was to compare the efficacy of a new bone graft substitute composed of a combination of mineralized and demineralized allograft, along with hyaluronic acid (AFT Bone Void Filler) with several other bone graft materials in a sheep vertebral bone void model. A drilled defect in the sheep vertebral body was filled with either the new DBM preparation, calcium sulfate (OsteoSet), autologous bone graft, or left empty. The sheep were euthanized after 6 or 12 weeks, and the defects were examined by histology and quantitative histomorphometry. The morphometry data were analyzed by one-way analysis of variance with the post hoc Tukey-Kramer test or the Student's t-test. All of the bone defects in the AFT DBM preparation group showed good new bone formation with variable amounts of residual DBM and mineralized bone graft. The DBM preparation group at 12 weeks contained significantly more new bone than the defects treated with calcium sulfate or left empty (respectively, p < 0.05, p < 0.01). There was no significant difference between the DBM and autograft groups. No adverse inflammatory reactions were associated with any of the three graft materials. The AFT preparation of a mixture of mineralized and demineralized allograft appears to be an effective autograft substitute as tested in this sheep vertebral bone void model.     

兔桡骨临界骨缺损模型的制备

背景：各种原因造成的骨折和骨缺损导致的骨不连一直是临床骨科修复的一大难题,相关产品质量的检验需要标准的骨缺损模型,然而骨缺损的临界长度至今仍无定论。 目的：建立兔桡骨骨缺损模型,以确定兔桡骨临界骨缺损长度。 方法：将18只雄性新西兰大白兔,随机分为6组,在双侧桡骨中段分别做12,13,14,15,16,17 mm的缺损,伤口缝合包扎但不固定。 结果与结论：大体标本和放射学观察显示,3个月内12,13,14,15,16 mm组的缺损均有修复完整的情况,但17 mm组无一例修复。组织学结果显示12,13,14,15,16 mm组缺损修复区有骨小梁与骨基质的形成、骨再生和再血管化、髓腔不同程度再通以及成骨细胞,17 mm组可见成骨细胞,破骨细胞,但未见骨再血管化和髓腔再通。故兔桡骨临界骨缺损长度为17 mm。     

Beta-TCP bone graft substitutes in a bilateral rabbit tibial defect model

The use of artificial bone graft substitutes has increased as the surgical applications widen and the availability of allograft bone decreases. The ideal graft substitute should reabsorb with time to allow and encourage new bone formation whilst maintaining its properties as an osteoconductive scaffold until it is no longer required. A potential disadvantage of some synthetic substitutes is their long dissolution time. Beta-tricalcium phosphates (beta-TCPs) have some advantages when compared to hydroxyapatite (HA), when used as a filler, in that it is more rapidly reabsorbed. Three commercially available and clinically used beta-TCP bone graft substitutes with the same chemistry (Vitoss, Osferion, Chronos) but with varying macro and microscopic characteristics were investigated using a bilateral tibial metaphyseal defect model in New Zealand white rabbits. When placed into tibial defects all three materials performed similarly in terms of mechanical properties of the healing defects. A decrease in properties was found at 12 weeks where implant resorption was nearly achieved while remodelling of the anteromedial cortex had yet to be completed. All materials were osteoconductive and supported new bone formation while implant resorption with time differed between materials. Vitoss resorbed faster than the other materials and is likely to differences in particle geometry, pore structure and interconnectivity.     

Cem-OsteticTM人工骨浆修复骨缺损

目的 Cem-OsteticTM人工骨浆为无机钙盐与纯净水混合物,可塑形、并可注射至骨缺损部位.观察Cem-OsteticTM人工骨浆修复四肢骨缺损临床疗效及不良反应.方法 2004年1月～2004年4月对10例10处骨缺损进行修复,年龄20～44岁,平均32岁.骨缺损范围为0.2cm×1cm～1cm×6cm.缺损的部位累及股骨、胫骨、肱骨、掌骨、锁骨.手术显露骨缺损部位后,修整骨断端,对失效内固定物予以更换,骨缺损部位植入Cem-OsteticTM人工骨浆.结果手术均获成功,术后随访5～7个月,平均为6个月.全部患者未见任何不良全身反应及局部反应,X线片显示固化后的Cem-OsteticTM人工骨浆与受区骨直接愈合,手术后测定血钙、磷值与术前对比无明显升高.结论 Cem-OsteticTM人工骨浆适用于四肢骨缺损修复.     

钛网打压植骨重建四肢长骨节段骨缺损☆

背景：四肢长骨节段骨缺损临床上常见,一直以来是骨科修复领域的难题。 目的：介绍钛网打压植骨重建四肢长骨节段骨缺损新方法的实验研究和临床用用。 方法：第一作者应用计算机检索1990至2011年 PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed), CNKI 数据库(www.cnki.net/index.htm), 和维普数据库(http//www.cqvip.com)有关钛网打压植骨重建四肢长骨节段骨缺方面的文献,英文检索词为“segmental bone defect,titanium mesh or titanium cage”;中文检索词为“四肢或长骨,骨缺损,钛网”。排除重复性研究、非四肢长骨和Meta分析类文献。根据纳入标准,共检索到30篇文献进行归纳总结。 结果与结论：钛网打压植骨重建四肢长骨节段骨缺损无论在大动物或是小动物的实验中均取得较好的成骨效果,优于非钛网的游离植骨和大块皮质骨游离植骨;自体骨、异体骨和人工骨均是可行的填充植骨材料,以自体松质骨为主的植骨材料的成骨效果相对较好,具有生物活性钛网较普通钛网的成骨效果更好。临床上,在四肢长骨各个部位的节段骨缺损中均取得很好的治疗效果,包括骨缺损长达十余厘米的病例。钛网打压植骨重建四肢长骨节段骨缺损新方法,改变了以往认为大于 6 cm以上长段骨缺损只适宜带血运的骨移植、而不适宜无血运游离植骨的传统观点。该方法较简单、安全、有效,是一种较理想的替代治疗长骨骨干部节段骨缺损的无血运游离植骨法。     

建立桡骨缺损模型：骨缺损标准尺寸的确立

背景：不同研究所采用的桡骨骨缺损模型长度均不同,目前尚没有统一的桡骨缺损标准尺寸。 目的：对兔桡骨缺损模型施行标准化研究再探讨,以期获得相对更可靠的兔桡骨缺损标准尺寸。 方法：选用健康6月龄新西兰大白兔40只,双侧桡骨共80侧,在麻醉下行兔桡骨手术造成桡骨完全骨膜缺损,将桡骨随机分为5组,分别为缺损1.0,1.2,1.4,1.7和2.0 cm组,每组16侧。分别于术后12周行X射线、大体解剖、CT三维重建和组织切片等检查,观察各组骨缺损愈合情况。 结果与结论：建模后12周可见缺损1.0 cm组至缺损2.0 cm组愈合率逐步下降,1.4 cm骨缺损组愈合率低于1.0及1.2 cm组(P < 0.05)。1.4,1.7,2.0 cm骨缺损组X射线评分及CT评分值均低于1.0及1.2 cm骨缺损组(P < 0.05),说明骨缺损尺寸在1.2 cm以下时,桡骨自行愈合率相对较高。结果提示实验成功建立了兔桡骨骨缺损模型,桡骨中段缺损(包括骨膜)尺寸≥1.4 cm更加可靠。     

Cem-Ostetic^TM人工骨浆修复骨缺损

目的Cem-OsteticTM人工骨浆为无机钙盐与纯净水混合物,可塑形、并可注射至骨缺损部位。观察Cem-OsteticTM人工骨浆修复四肢骨缺损临床疗效及不良反应。方法2004年1月~2004年4月对10例10处骨缺损进行修复,年龄20~44岁,平均32岁。骨缺损范围为0.2cm×1cm~1cm×6cm。缺损的部位累及股骨、胫骨、肱骨、掌骨、锁骨。手术显露骨缺损部位后,修整骨断端,对失效内固定物予以更换,骨缺损部位植入Cem-OsteticTM人工骨浆。结果手术均获成功,术后随访5~7个月,平均为6个月。全部患者未见任何不良全身反应及局部反应,X线片显示固化后的Cem-OsteticTM人工骨浆与受区骨直接愈合,手术后测定血钙、磷值与术前对比无明显升高。结论Cem-OsteticTM人工骨浆适用于四肢骨缺损修复。     

医用硫酸钙人工骨与同种异体骨修复良性骨肿瘤骨缺损的对比分析

背景：探讨医用硫酸钙人工骨可替代同种异体骨作为骨缺损的修复材料的可行性。 目的：观察医用硫酸钙人工骨材料治疗良性骨肿瘤骨缺损的临床疗效。 方法：纳入应用医用硫酸钙人工骨材料于临床修复良性骨肿瘤刮除术后骨缺损病例31例和同种异体骨材料修复病例36例。观察植入材料后4,8,12周摄X射线片植骨吸收情况及植骨材料降解率。 结果与结论：全部病例均获随访3个月以上。两组病例在随访期间植骨材料降解率差异无显著性意义。良性骨肿瘤植骨区无复发。患者植入材料后3个月X射线片示骨缺损部位有新骨生成。结果表明医用硫酸钙人工骨材料临床疗效和同种异体骨接近。     

Bi-Ostetic^TM人工骨粒修复四肢骨缺损

目的 Bi-OsteticTM人工骨粒为羟基磷灰石(HAP)与磷酸钙(TCP)混合人工骨材料,观察Bi-OsteticTM人工骨粒修复四肢骨缺损临床疗效及不良反应。方法 2004年1月-2004年4月对12例12处骨缺损进行修复,年龄20-59岁,平均39岁。骨缺损范围为0.4cm×1cm-1cm×5cm。缺损的部位累及股骨、胫骨、肱骨、掌骨、锁骨。手术显露骨缺损部位后,修整骨断端,重新更换内固定物后,植入Bi-OsteticTM人工骨粒。结果 手术均获成功,术后随访4-7个月,平均为5个月。全部患者未见任何不良的全身反应及局部反应,X线片显示Bi-OsteticTM人工骨粒与受区骨愈合良好,12例术后测定血钙、磷值无明显升高。结论Bi-OsteticTM人工骨粒适用于四肢骨缺损修复。     

Bi-OsteticTM人工骨粒修复四肢骨缺损

目的 Bi-OsteticTM人工骨粒为羟基磷灰石(HAP)与磷酸钙(TCP)混合人工骨材料,观察Bi-OsteticTM人工骨粒修复四肢骨缺损临床疗效及不良反应.方法 2004年1月～2004年4月对12例12处骨缺损进行修复,年龄20～59岁,平均39岁.骨缺损范围为0.4cm×1cm～1cm×5cm.缺损的部位累及股骨、胫骨、肱骨、掌骨、锁骨.手术显露骨缺损部位后,修整骨断端,重新更换内固定物后,植入Bi-OsteticTM人工骨粒.结果手术均获成功,术后随访4～7个月,平均为5个月.全部患者未见任何不良的全身反应及局部反应,X线片显示Bi-OsteticTM人工骨粒与受区骨愈合良好,12例术后测定血钙、磷值无明显升高.结论 Bi-OsteticTM人工骨粒适用于四肢骨缺损修复.     

幼龄兔及成年兔桡骨骨缺损模型中骨缺损大小的对比研究

目的 探讨不同月龄新西兰兔建立桡骨骨缺损模型时骨缺损大小的选择。方法 选取3月龄(幼龄兔)及6月龄(成年兔)健康雄性新西兰兔各20只,根据兔龄分为A组(3月龄)和B组(6月龄),每组兔前肢采用数字表法随机分为2亚组制备桡骨骨缺损模型,每组20侧。其中A1组、B1组桡骨骨缺损长度为15 mm,A2组、B2组骨缺损长度为20 mm。分别于模型制备术后第4、8、12周行X线检查,并应用X线Lane-Sandhu评分标准评估骨愈合情况。术后12周处死所有实验动物,取桡骨标本进行大体及组织学观察,分析骨愈合情况。结果 制备骨缺损模型术后,所有实验兔均存活。X线检查显示:术后第8周A1组骨缺损基本愈合,至第12周新生骨塑形完全,与正常桡骨形态类似;其余3组至第12周骨缺损均未完全修复,断端及邻近尺侧有少量新骨生成,髓腔封闭。术后各时间点X线Lane-Sandhu评分结果示:组内比较,A1组评分均高于A2组,差异均有统计学意义(P值均<0.05);B1组与B2组评分比较,差异均无统计学意义(P值均>0.05)。组间缺损尺寸相同的亚组间比较:A1组、A2组评分分别高于B1组、B2组,差异均有统计学意义(P值均<0.05)。术后12周标本大体观察显示:A1组断端形成骨性桥接,新生骨塑形良好,其余3组断端髓腔封闭,缺损区由纤维组织填充。组织学结果示A1组修复完全,新生骨骨板排列规则;其余3组缺损空腔可见纤维组织填充。结论 兔龄和骨缺损大小对于术后骨缺损愈合情况有重要影响,在构建兔桡骨骨缺损模型的动物实验中,幼龄兔(3月龄)骨缺损长度宜选择20 mm,成年兔(6月龄)宜选择15 mm。     

A model for tissue engineering applications: femoral critical size defect in immunodeficient mice

Animal models for preclinical functionality assays lie midway between in vitro systems such as cell culture and actual clinical trials. We have developed a novel external fixation device for femoral critical size defect (CSD) in the femurs of immunodeficient mice as an experimental model for studying bone regeneration and bone tissue engineering. The external fixation device comprises four pointed rods and dental acrylic paste. A segmental bone defect (2 mm) was created in the midshaft of the mouse femur. The CSD in the femur of the mice were either left untreated or treated with a bone allograft, a cell-scaffold construct, or a scaffold-only construct. The repair and healing processes of the CSD were monitored by digital x-ray radiography, microcomputed tomography, and histology. Repair of the femoral CSD was achieved with the bone allografts, and partial repair of the femoral CSD was achieved with the cell scaffold and the scaffold-only constructs. No repair of the nongrafted femoral CSD was observed. Our results establish the feasibility of this new mouse femoral model for CSD repair of segmental bone using a simple stabilized external fixation device. The model should prove especially useful for in vivo preclinical proof-of-concept studies that involve cell therapy-based technologies for bone tissue engineering applications in humans.     

Histologic and morphometric study of chronic gastritis in alcoholic patients.

The effect of chronic alcohol ingestion on the gastric mucosa was determined in 96 patients in whom gastroscopy was performed because of upper gastrointestinal symptoms. Seventy-two patients were classified as alcoholics and 24 patients as nonalcoholics. In all cases biopsy specimens were taken from the fundus and antrum. The diagnoses of chronic superficial and atrophic gastritis were based on conventional histologic criteria and a morphometric technique utilizing quantitation of the chronic inflammatory cells in the lamina propria. Alcoholic patients were found to have a higher incidence of chronic gastritis of the antrum than nonalcoholics (p less than 0.001). Fundic involvement was also more common, probably accounting for the decreased gastric acid output described in chronic alcoholic patients. Finally, gastritis was more severe in the alcoholics; below 45 years of age chronic atrophic gastritis was seen only in alcoholics. We conclude that chronic gastritis develops more frequently in alcoholic patients and evolves into chronic atrophic gastritis at an earlier age than in nonalcoholic subjects.     

Quantification and morphometric analysis of vascular structures in the bone marrow. A critical review

The important role of angiogenesis for invasion, tumor growth, metastasis and progression of solid tumors is well established. Only recently, the clinical significance of neovascularization in hematological neoplasms has come into the focus of scientific efforts. Emerging data from these studies suggest that the vascular architecture exerts a pivotal role in acute leukemias and multiple myeloma as well as myeloproliferative disorders. Thus, angiogenesis seems to maintain a general condition for malignant growth and the progression of hematological diseases. However, a major shortcoming is that most of the previous studies include only a simple quantitative evaluation of the microvessel density, and applied methods which were developed primarily for the characterization of angiogenesis in solid tumors. In this context it should not be overlooked that solid neoplasms and the bone marrow reveal significant differences regarding the vascular structures and the distribution of microvessels. Therefore, the usually applied, so-called hot-spot techniques, should be amended by an appropriate and more elaborate computer-assisted morphometric analysis of the microvessel structures. This additional spectrum of information regarding quantity and quality of angiogenesis, enables a further understanding of the morphological changes in the course of diseases, and in particular the effect of various therapies on bone marrow vascularization.     

锥形束CT数字成像分析牙槽嵴裂植骨修复的成骨效果

BACKGROUND:In alveolar cleft patients, the amount of bone stock after alveolar bone grafting is mostly measured and analyzed by two-dimensional imaging, which can result in a large error.OBJECTIVE:To evaluate the 6-month osteogenesis of alveolar bone graft in alveolar cleft patients using cone beam CT.METHODS: Alveolar bone grafting was performed in 25 patients with unilateral complete alveolar cleft. The patients were followed up for 6 months after surgery and the osteogenesis of the bone graft was evaluated by CBCT.RESULTS AND CONCLUSION: After the surgery, the labial bone support was better than the palatal one. There were significant differences in the alveolar bone thickness of the cleft region and the normal region of the central incisor as well as the alveolar bone thickness of the cleft region and the normal region of the canine tooth 0 mm distant to the alveolar crest. These findings indicate that the palatal bone support is less than the labial one, and the bone support of the central incisor is not satisfactory, which provide the basis for the tooth movement in the alveolar bone grafting and the orthodontics 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     

Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects

The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing a CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100 and 500 ng ml⁻¹) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2 and 5 μg) on bone formation was evaluated using rat critical size segmental bone defects for 4 weeks. Fourier transform infrared spectroscopy spectra and scanning electron microscopy images showed chemical and structural changes by the addition of fibrinogen into the chitosan-lactide copolymer. The incorporation of fibrinogen molecules significantly increased the compressive modulus of the hydrogels. The in vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4 weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiography, microcomputed tomography and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration.