冷冻异体骨膜引导骨组织再生的实验研究

目的：探讨用冷冻异体骨膜作为引导骨组织再生膜的可能性；方法：取兔颅骨顶部骨膜经冷冻处理，在３１只日本大耳白兔的下颌骨造成两处骨缺失，采取同体对照，一处表面覆盖冷冻异体骨膜，另一处不覆盖。分别在第４、８、１２、１６周处死动物，行Ｘ线及组织学观察。结果：冷冻异体骨膜不产生排斥反应，可在体内维持８￣１２周，具有良好的阻挡纤维组织长入骨创面、分隔不同细胞及引导组织再生的功效。结论：冷冻异体骨膜是一种理想的     

Influence of platelet-rich plasma on bone regeneration: a histomorphometric study in rabbit calvaria

PURPOSE: The aim of this study was to evaluate whether local application of platelet-rich plasma (PRP) would induce bone regeneration in cranial defects on rabbits. MATERIALS AND METHODS: Twelve female New Zealand rabbits were used for this study. Two identical 10-mm-diameter bicortical cranial defects were created in each animal. One of the defects was grafted with PRP, while the contralateral was left unfilled as a negative control. Animals were sacrificed at 2, 4, 6, and 8 weeks after surgery, and biopsy specimens were evaluated histologically and histomorphometrically under light microscopy. Analysis of variance was used for statistical analysis. RESULTS: The histomorphometric evaluation showed more regenerated bone after local administration of PRP at 2 weeks (P > .05), 4 weeks (P < .05), and 6 weeks (P > .05). At week 8, new bone formation was comparable in both groups. CONCLUSION: In this animal model, local application of PRP in bone defects enhances healing significantly at 4 weeks.     

A multidisciplinary approach to the healing of cranial and residual maxillary cleft defects by means of allogenous demineralized osseous implants and polylactic acid casts in dogs.

OBJECTIVE: The aim of this study was to evaluate the healing of artificially induced bony defects in dogs by means of demineralized allogenous bone powder (DBP) implants covered with polyhydroxy lactic acid (PLA) casts compared with DBP implants without the casts. DESIGN: Prospective animal study. SETTING: Research university. SAMPLE: Following a pilot study in which two dogs were used, four mongrel dogs between the ages of 18 and 24 months and weighing approximately 20 kg were used as subjects. INTERVENTIONS: Each experimental animal had bilateral maxillary alveolar clefts created. In a later procedure, each defect was repaired with a DBP implant, half of which were covered with a PLA96 matrix. Each animal also had a circular defect created in each parietal bone that was immediately covered with DBP implants, half of which were similarly covered with a PLA96 disk. MAIN OUTCOME MEASURE: Repeated technetium-99m methylene-diphosphate (99mTC MDP) uptake measurements were performed to evaluate bone metabolism during the healing period, while at relevant intervals, radiographs were taken of the healing alveolar cleft defects to register bone repair. After 1 year, the animals were euthanized for macroscopic and histologic evaluation. RESULTS: Histologically, the grafts covered with PLA96 were at a more advanced stage of healing than those without, and the cranial defects similarly were more advanced in the healing process than the alveolar defects. Uptake of 99mTC MDP into the cranial implants was at its maximal level after 1 week and then gradually decreased until, after 7 weeks, it was not significantly different from zero. Cranial defects covered with a PLA95-enhanced implant showed a mean maximum count rate of 275, while the plain DBP side showed a mean maximal count rate of 150. Alveolar defects with the plain DBP implant showed a maximum count rate in the first week; those with the PLA95 enhanced implant showed maximum uptake during the second week. On both sides, there was a gradual decrease to the base value in the seventh week. The mean maximum count on the PLA96-enhanced side was 285, while on the plain DBP side, the corresponding value was 320. CONCLUSION: Although an advantage of the combination was established for parietal cranial defects, no advantage was seen for alveolar cleft defects in this experimental setup.     

Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits

The reconstruction of large bony defects remains a clinical challenge, and angiogenesis and neovascularisation are being given more attention in bone tissue engineering. In this study we cocultured peripheral blood CD34+ cells (PB-CD34+ cells), an endothelial progenitor cell/haematopoietic stem cell-enriched population, with bone marrow-derived mesenchymal stem cells (MSC) to investigate their potential for bony regeneration. Cocultured cells showed better osteogenic differentiation than MSC alone in vitro. The cocultured cells and MSC sheets were also composited with hydroxyapatite and implanted in calvarial critical-size defects in rabbits. The rabbits were killed before microcomputed tomographic (MicroCT) and histological analysis. The results showed that cocultured cell composites had promoted bony regeneration more efficiently by 8 weeks after implantation. Our results indicate that the coculture of PB-CD34+ cells and MSC increases bony regeneration in calvarial critical-size defects in rabbits, and provide a new promising therapeutic strategy to aid skeletal healing.     

Acceleration of de novo bone formation with a novel bioabsorbable film: a histomorphometric study in vivo

Objective:  Different types of barrier membranes have been used in periodontal applications for the technology of guided tissue regeneration (GTR). The aim of this study was to characterize the biological effect of novel calcium alginate film (CAF) on bone tissue regeneration by using rabbit mandible defects model.
Methods:  A critical size defect (5 mm in diameter) was created in the bilateral corner of mandible of 45 adult rabbits. The defects were covered with CAF served as the experimental group, or conventional collagen membrane (CCM) or left empty as the controls. Animals were killed after 1, 2, 4, 6 and 8 weeks. Morphological and histomorphometric studies were performed to evaluate their bone regeneration pattern and biological effects.
Results:  Histological sections showed that bone regeneration pattern was centripetal in growth from defect rim. The quantitative histometry analysis revealed a significantly greater percentage of newly generated bone in CAF defects than that in CCM defects and empty defects from 2 to 6 weeks post-operation ( P  < 0.01). After 6 and 8 weeks, significantly more mature lamella bone had formed with CAF than with CCM. Empty control defects showed bone formation starting from the defect margins and incomplete healing even after 8 weeks.
Conclusion:  The CAF guided early bone growth and appeared more effective as a bioabsorbable GTR membrane than CCM. This study with mandible defect model suggests that bone defects augmented with CAF may offer most promising results from a histological and histomorphometric perspective.     

脂肪干细胞复合成骨性干细胞膜片用于修复兔颅骨缺损的实验研究

目的　细胞膜片技术已经被证实了可以成功用于多种组织再生,我们前期实验证明了脂肪干细胞（ADSCs）可以显著促进成骨性细胞膜片（BMSCs sheet）的异位成骨能力。因此,本实验的目的是验证ADSCs能否促进成骨性BMSCs膜片的骨缺损修复能力。方法：ADSCs和BMSCs分别取自同一个供体的腹股沟和髂骨骨松质,通过连续成骨诱导的方法制备成骨性BMSCs膜片,并分析BMSCs膜片的特性;然后将ADSCs复合在BMSCs膜片上,构架ADSCs-BMSCs膜片复合体,移植到兔颅骨缺损模型,单纯BMSCs膜片和颅骨缺损组作为对照。移植八周后,Micro-CT扫描和组织学分析其骨修复再生情况。结果：BMSCs膜片由含有大量矿化结节的多层细胞膜片构成;移植八周后,相比于对照组,实验组新生组织骨密度更高,新生矿化组织量也更多,同时新生骨组织/总新生组织体积（BV/TV）的比值也更高,表明ADSCs显著提高了BMSCs膜片的骨缺损修复能力。结论：ADSCs复合BMSCs膜片可以明显促进兔颅骨缺损的骨再生修复,该方法为促进骨修复重建提供了一种新的方法。     

国产非降解生物膜引导动物颅骨组织再生的组织学观察

目的 :观察两种国产非降解生物膜引导组织再生的组织学变化 .方法 :选用兔颅骨开窗缺损模型覆盖两种非降解生物膜 ,空白对照 ,术后 2、4、8、12周取材 ,常规制片 ,光镜、透射电镜、扫描电镜观察 .结果 :两种国产非降解生物膜有较好的组织相容性 ,对骨缺损愈合有保护和促进作用 .结论 :临床使用国产非降解生物膜引导组织再生是可行的 .     

Regeneration of the sagittal suture by GTR and its impact on growth of the cranial vault

The aim of this study was to investigate the effect of bone grafting, suture transplantation, and guided tissue regeneration (GTR) treatment on healing of craniectomy defects involving the sagittal cranial suture and on the growth of the cranial vault. Fifty 4-week-old rats were included in the study. A 5.0 mm wide trephine defect was created with its midline corresponding to the sagittal cranial suture between the coronal and occipital cranial vault sutures. The animals were randomly allocated to five groups of 10 animals. Group A: The cranial defect was left untreated. Group B: An occipital bone graft was placed into the cranial defect. Group C: A cranial bone graft including a portion of the frontal suture was placed in the cranial defect. Group D: The cerebral and galeal aspect of the defect were covered with an e-PTFE membrane. Group E: The animals were sham-operated, no defect was created. In all animals, two gutta-percha points were placed demarcating the lateral borders of the parietal bones. Histological analysis at 4 months following surgery showed that the untreated cranial defects healed with fibrous connective tissue. The bone-grafted defects healed partially with bone and connective tissue in the periphery of the bone graft. The healing of suture-grafted defects resembled that of the bone-grafted defects, since the transplanted suture became completely obliterated with bone. The membrane-treated defects healed with bone and a suture-like tissue resembling the sagittal suture of the sham-operated controls. Cephalometric measurements demonstrated that membrane-treated and sham-operated control animals exhibited significantly more (P < 0.05) coronal growth (approximately 1.2 mm) than that of the remaining three groups of animals (approximately 0.7 mm). These findings were supported by the craniometry measurements demonstrating that sham-operated control and membrane-treated specimens presented significantly more cranial width than that of the remaining groups of animals (P < 0.05). It can be concluded that complete osseous healing, creation of a new sagittal suture, and increased cranial growth can be achieved by the treatment of craniectomy defects with the GTR technique.     

Closure of palatal defects without a surgical flap: an experimental study in rabbits.

PURPOSE: This study evaluated the use of resorbable calcium sulfate with and without bone grafting in palatal defects in rabbits as a guide to regeneration of the mucoperiosteal tissue and bone. METHODS AND MATERIALS: Eighteen New Zealand rabbits were used in the experiment. A 1-cm wide region of mucoperiosteum, nasal mucosa, and bone between the incisor and premolar teeth was excised from the left side of the palate to create a defect. These gaps were left open and unfilled in 6 animals as a control (group 1). Defects in a second group of 6 animals were packed with calcium sulfate (group 2). The osseous defects in a third group of 6 animals were filled with bovine demineralized xenographic bone particles, and the soft tissue gaps were covered with calcium sulfate (group 3). The various sites were evaluated clinically at 1, 3, and 4 weeks, and then at 3 months after surgery. All animals were killed at the 3 months period, and the sites were evaluated histologically. RESULTS: The calcium sulfate had resorbed and the mucoperiosteal margins of the defects in both experimental groups had regenerated and the soft tissue gaps were closed at 4 weeks. Osseous defects in group 3 showed complete bone regeneration compared with osseous defects in group 2. Defects in the control group showed persistent oronasal fistulae and fibrous healing. CONCLUSION: Open mucoperiosteal wounds in rabbits packed with calcium sulfate can heal uneventfully, and the gaps can be closed successfully.     

Calcium alginate film used for guided bone regeneration in mandible defects in a rabbit model

The objective of this research was to study a new bioabsorbable membrane material, calcium alginate film (CAF), used for guided tissue regeneration (GTR) or guided bone regeneration (GBRL). Circular bone defects of five mm diameter were created in the corners of the mandibles in 45 rabbits. The defects covered with calcium alginate film (CAF) served as the experimental sites, and collagen membrane (CM) or no membrane served as the control sites without considering left or right side, just with a mark on the ear of the same side. The healing condition was analyzed by histological studies and histometry analysis after one, two, four, six, and eight weeks. The histological evaluation showed that the bone regeneration pattern was centripetal in growth from the defect rim. The quantitative histometry analysis showed significantly more and faster newly generated bone in CAF defects than that in CM defects or in empty defects (p < 0.01) at two, four, six, and eight weeks postsurgically. Calcium alginate film was more effective for GTR and GBR than the collagen membrane.     

胚胎骨引导骨再生膜下植入对颌骨缺损修复的意义

目的 评价胚胎骨（Fetal BOne,FB)的空间维持能力和聚乳酸膜（PLA）胚胎骨复合植入（PLA／FB）的骨修复效果。方法 在18只日本大耳白兔双侧下凳骨下缘形成方形骨缺损，一侧缺损表面以PLA膜严密覆盖，另一侧缺损内植入FB后表面覆盖PLA膜，术后4，8，12周分期处死动物，标本行大体观察，X线检查及组织学观察，结果 术后各期单纯PLA膜侧多数标本发生程度不同的膜塌陷，复合植入侧仅3例发生轻度膜陷，组织学观察，术后4，8周复合植入侧成骨量高，骨组织丰富，12周时复合植入侧再生皮质骨连续致密。结论 PLA／FB复合移植修复骨缺损成骨活性高，骨量多，骨外形恢复优于单纯PLA膜侧。     

Guided bone regeneration of cranial defects, using biodegradable barriers: an experimental pilot study in the rabbit.

The aim of this study was to test if a biodegradable barrier could be used to achieve proper bone healing of full-thickness trephine skull defects, applying the biological principle of guided tissue regeneration (GTR). Two New Zealand white rabbits were used. In each animal, 2 circular through-and-through bone defects with a diameter of 8 mm were created in the midline of the frontal and parietal bones of the calvarium. One defect was covered with the mucoperiosteal flaps without placement of an intervening membrane barrier (control). One test defect (test 1) was covered by a biodegradable, non-porous polylactic acid membrane on the outer (supra-calvarial) side of the defect, and 2 test defects (tests 2 and 3) were covered by similar membranes on both the outer and the inner aspects of the defects, prior to flap closure. 6 weeks postsurgically, the animals were sacrificed and the defect areas including surrounding tissues were harvested for histological preparation. The control defect was essentially occupied by supra-calvarial soft tissue, located in direct contact with the dural tissue. In the test cavities, there was a continuous bridge of regenerated bone extending from one edge of the defect to the other, although in test 1 not attaining the same thickness as the bone bordering the defect. In the 2 other test defects, the regenerated bone had reached a thickness almost corresponding to that of the surrounding bone. The bone regeneration was achieved without recourse to adjunctive bone graft materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incomplete bone regeneration of rabbit calvarial defects using different membranes

The present study describes the use of a degradable and a non‐degradable material for guided bone regeneration. Forty rabbits were divided into 5 groups. Bicortical defects 15 mm in diameter were prepared in rabbit calvaria. A titanium microplate was placed over the defect to prevent collapse of the membrane. The calvarial defects of 2 groups were covered by an outer expanded polytetrafluoroethylene (e‐FTFE) membrane respectively by a Polyglactin 910 membrane. Bicortical e‐PTFE membranes or Polyglactin 910 membranes were used in 2 other groups. The defects were not covered by membranes in the control group. Undecalcified sections were prepared for histologic evaluation after an observation period of 8 weeks. Complete bone healing of the defects was not observed in any of the specimens. The Polyglactin 910 material lacks physical strength, resulting in collapse of the membrane and brain tissue hemiation into the defects. Subsequently, bone regeneration was impaired. The cellular reactions due to degradation of the material were minor and did not interfere with bone healing. Defects covered bicortically by e‐PTFE membranes revealed the largest amount of regenerated bone. The e‐PTFE membrane induced a severe cellular reaction, but no inhibition of bone regeneration was noted.     

Heilungsverhalten und Knochenregeneration von maxillären Osteotomien im experimentellen Kaninchenmodell

Purpose: The development of fibrous nonunions following orthognathic surgery is thought to result from an interaction of biomechanical stress and the differential and more rapid migration of fibroblasts compared to osteoblasts into the wound site during healing. The present study was designed to test this hypothesis through the manipulation of guided tissue regeneration and osteotomy fixation techniques in an experimental rabbit model. Materials and methods: Bilateral critical size (4 mm) defects ¶(n = 24) were produced in the maxillae of 12 adult New Zealand White rabbits. The maxillary segments were rigidly or nonrigidly fixed using bone microplates and screws or osteosynthetic wires. The defects were then covered with a resorbable collagen membrane or left uncovered. The rabbits were followed for 4 weeks with serial dorsoventral and lateral oblique cephalographs and the maxillae were then harvested for histological analysis. Results: Radiographic and histomorphometric analysis revealed that rigidly fixed defects, covered with membrane, showed the most rapid and organized new bone formation. The rigidly fixed defects with membrane averaged approximately 40% more new bone in the osteotomy site than the rigidly fixed defects with no membrane. Nonrigidly fixed defects with no membrane also showed an ingrowth of fibroblasts and fibrous nonunions. Conclusion: These results suggest that an interaction between the decreased fibrous tissue ingrowth through guided tissue regeneration and osteotomy segment stability from rigid fixation prevented postoperative fibrous nonunions and facilitated new bone regeneration and osteotomy site healing in this rabbit model.     

Effects of fixation type and guided tissue regeneration on maxillary osteotomy healing in rabbits.

PURPOSE: The development of fibrous nonunions after orthognathic surgery is thought to result from an interaction of biomechanical stress and the differential and more rapid migration of fibroblasts (compared with osteoblasts) into the wound site during healing. The present study was designed to test this hypothesis through the manipulation of guided tissue regeneration and osteotomy fixation techniques in an experimental rabbit model. MATERIALS AND METHODS: Bilateral critical size (4 mm) defects (n = 24) were produced in the maxillae of 12 adult New Zealand white rabbits. The maxillary segments were rigidly or nonrigidly fixed using bone microplates and screws or osteosynthetic wires. The defects were then covered with a resorbable collagen membrane or left uncovered. The rabbits were followed for 4 weeks with the use of serial dorsoventral and lateral oblique cephalographs, and the maxillae were then harvested for histologic analyses. RESULTS: Radiographic and histomorphometric analyses revealed that rigidly fixed defects, covered with membrane, showed the most rapid and organized new bone formation. The rigidly fixed defects with the membrane averaged approximately 40% more new bone in the osteotomy site compared with the rigidly fixed defects with no membrane. Nonrigidly fixed defects with no membrane showed an ingrowth of fibroblasts and fibrous nonunions. CONCLUSIONS: These experimental results suggest that an interaction between the decreased fibrous tissue ingrowth through guided tissue regeneration and osteotomy segment stability from rigid fixation prevented postoperative fibrous nonunions and facilitated new bone regeneration and osteotomy site healing in this rabbit model.     

Unbiased stereological methods used for the quantitative evaluation of guided bone regeneration

The present study describes the use of unbiased stereological methods for the quantitative evaluation of the amount of regenerated bone. Using the principle of guided bone regeneration the amount of regenerated bone after placement of degradable or non-degradable membranes covering defects in rabbit calvaria was compared. Forty rabbits were divided into 5 groups. A titanium microplate was placed over the defect to prevent collapse of the membrane. The non-degradable expanded polytetrafluoroethylene membrane and the degradable Polyglactin 910 material were both placed unicortically and bicortically. Undecalcified sections were prepared for stereologic evaluation after an observation period of 8 weeks. Complete bone healing of the defects was not observed in any of the specimens. Unbiased stereologic estimates revealed 48% bone regeneration in defects covered by 2 ePTFE membranes, and 12% in defects covered by 2 Polyglactin 910 membranes. Defects covered by 1 ePTFE or 1 Polyglactin 910 membranes revealed 10% or 18% bone regeneration, respectively. The control group regenerated 14%. The major difference of the estimates was caused by real difference between specimens, i.e. biologic variation, whereas only minimal variance was added by the stereologic estimation procedure.     

Bone repair of experimentally induced through-and-through defects by Gore-Tex, Guidor, and Vicryl in ferrets: a pilot study

OBJECTIVE: The purpose of this pilot study was to evaluate whether improved bone regeneration can be achieved in experimentally induced through-and-through mandibular defects in ferrets and whether there is a quantitative and qualitative difference of regenerated bone with different guided tissue regeneration membranes. STUDY DESIGN: Through-and-through osseous defects were created at the apices of 16 mandibular premolars of 8 ferrets. The transosseous defects were covered with Gore-Tex, Vicryl, or Guidor membranes. As control, the defects were covered with mucoperiosteal flaps without any membranes. RESULTS: The control group showed ingrowth of sulcular epithelium into the defect. The Gore-Tex group showed good bone formation, whereas the Vicryl group showed the highest degree of bone formation. Six weeks after the operation, the defect had nearly completely filled with fibered and lamellar bone. Twelve weeks after the operation, mainly lamellar bone was observed. In contrast, the Guidor group was found to have limited bone regeneration. CONCLUSION: The results of this in vivo study suggest that guided tissue regeneration membranes generally promote and improve bone regeneration in osseous defects of endodontic origin.     

纯钛膜治疗颅骨局部骨缺损的动物实验研究

目的：评价国产无孔纯钛膜引导兔顶骨区局部骨缺损的骨再生修复效果。方法：在8只大白兔的左右顶骨区建立骨缺损，一侧覆盖钛膜，另外一侧作为对照侧。随机分成4周组和8周组。进行肉眼、直接数字化X线影像及硬组织切片组织学观察。结果：对照侧骨缺损的面积减小、但中央区缺损未愈合，纤维组织修复；而实验侧骨缺损均骨性愈合。结论：钛膜有非常好的生物相容性，阻挡软组织长和骨缺损区内，并较好维持骨再生修复空间，保证骨缺损修复。     

Endothelial progenitors enhanced the osteogenic capacities of mesenchymal stem cells in vitro and in a rat alveolar bone defect model

ObjectiveTransplantation of mesenchymal stem cells (MSCs) may promote bone healing. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of MSCs by improving their microenvironment. We aimed to investigate whether EPCs can enhance the osteogenic properties of MSCs in vitro, and whether transplantation of EPC-MSC cell sheets could promote bone regeneration in a rat model of alveolar bone defect.DesignMSCs and EPCs were obtained from 2-week-old Sprague-Dawley rats. Cell sheets were prepared using MSCs and MSCs co-cultured with EPCs. Morphological characteristics of cell sheets were observed by H&E staining. Osteogenic differentiation capacities of the cell sheets were assessed by alkaline phosphatase (ALP) staining, Alizarin Red S staining and qRT-PCR. Cell sheets were transplanted into alveolar bone defects in 8-week-old rats. Six weeks later, bone formation was assessed by micro-CT.ResultsEPC-MSC sheets exhibited faster osteogenesis than MSC sheets. Six weeks after implantation, alveolar bone defects transplanted with EPC-MSC sheets exhibited a better bone reconstruction. MSC sheets generated new bone that partially covered the defect areas, while EPC-MSC sheets exhibited more robust osteogenic activity, with continuous new bone that almost covered the entire defect area.ConclusionsTransplantation of cell sheets containing EPCs and MSCs promoted bone regeneration.