Application Study Of Collagen Membrane Combined With Bone Growth Stimulated Factors In Repair Of Mandibular Defect

Preface: Maxilla and mandible are the more complicated bones of human body which located at the middle and inferior part of cranio-maxillo-facial region, and mandible is the only bone which can move among all cranio-maxillo skeleton. They take part in many important functions such as construction of the outline of inferior half part of the face, manducation, occlusion, speaking, and keeping the air tube smooth. Some congenital or acquired factors may lead to segmental defect or dissymmetry of jaw bones which usually results in severe functional incapacitation and facial deformity. Repair of cranio-maxillo-facial defect aims at reconstruction of the integrity of jaw bone, recovering its function and correcting the facial deformity. There are many methods to repair the cranio-maxillo-facial deformity, and the main traditional method is bone transplantation which consists of autogenous bone transplantation, xenoma or heterogenous bone transplantation and artificial bone substitute. Autogenous bone transplantation has positive curative effect and has been used for a long time because it possesses many advantages, such as remaining the function of bone conduction and bone induction, containing osteoblasts, without risk of transmitting diseases, and increasing the anti-infection ability of the bone graft by vascular anastomosis or pedicle skin flap bone transplantation. But it still can't achieve an ideal repair, because of lack of donator, difficulty in shape carving, and auto damage. The concept of guided tissue regeneration (GTR) has been put forward in recent years. As a kind of technique applied in treatment of periodontal disease in stomatology, its principle is that separating different tissues by machinery barrier function in order to build an advantageous environment which could make the given tissue exert regeneration function mostly and reach the purpose of directional tissue repair. Acting as a new type of bone regeneration technique, it can enhance the regeneration capacity of bone tissue to some extent, accelerate the metabolism of bone tissue, avoid fabric healing of bone defect, and promote synostosis. This technique establishes a new treatment territory for bone defect. Our research group has accumulated rich experience of operation on mandible and has acquired satisfactory facial outline by years of operation on changing facial form and repairing excessive-bone-removal of mandibular angle. These experience provides some academic evidence for our experiment. We improved the method of autogenous bone transplantation on mandibula and got satisfactory result, by means of filling defect area with grain bone covered by collagen membrane combined with bone growth stimulated factors to reconstruct mandible shape through intraoral approach.Objectives: To identify the mechanism of ossification and agglutination of guided tissue regeneration (GTR) technique, we designed an animal model of segmental mandibular defect which was repaired with autogenous grain bone transplantation covered by collagen membrane combined with bone morphogenetic protein 2, and detected ossification indexes subsequently. Through the experiment we can understand the mechanism of ossification and test the effect of ossification so as to seek an easy-operated and effective method to repair segmental mandibular defect. Materials and Methods: The research was carried out through both animal experiment and clinical application. The main contents of animal experiment are composed of setting up the mould of segmental mandibular defect and repairing it with autogenous grain bone transplantation covered by collagen membrane combined with bone morphogenetic protein 2 and detecting ossification indexes subsequently. As experiment objects, 30 New Zealand rabbits were caused segmental mandibular defect by grinding osteotomy. We chose randomly one side of mandibula on which to do the transplantation with autogenous grain bone covered by collagen membrane combined with bone morphogenetic protein 2 as the experiment group, the other side on which to do nothing as the control group. Self-comparison was made on all objects. The rabbits were killed at 2, 4, 8, 12 and 24 weeks after operation respectively, then we tested corresponding ossification indexes from the following 3 aspects: 1. Gross sample, X-ray and histology detection. The ossification effect of the two groups were compared by observation of the gross sample and X-ray detection after obtaining the integrated mandibula sample, then ossification procedure was analyzed by observing the HE-dyeing histology sections under light microscope. 2. Observation of ultrastructure of ossification. By observing the ultrastructure changes of the reconstruction of bone defect, we can make sure the ossification mechanism. 3. Quantitative detection of ossification. Before being killed, the rabbits were labelled with fluorescence. After getting the sample, we detected the bone density, then took the data of 4 weeks, 12 weeks that including the region near the defect center and other defect area, and 24 weeks postoperatively as parameters. By statistics analysis, we can understand the tiny morphological changes of the new bone tissue during the bone healing. Clinical application: The clinical subjects involved 7 patients ( including 1 male and 6 female who age from 20-35) with segmental mandibular defect who had been treated by our department in recent 2 years. Among the patients, 4 cases were caused by excessive-bone-removal and 3 cases is congenital osteodysplasty. We tested the feasibility of this method and estimated the curative effect according to the following 2 aspects: 1. The directive significance of 3D spiral CT re-establishment for reconstruction of clinical cranio-maxillo-facial defect. The preoperative and postoperative 3D spiral CT re-establishment can provide imageology direction for mandibular defect reconstruction and check ossification effect. 2. Clinical application manifestation of repairing cranio-maxillo-facial defect with guided tissue regeneration (GTR) technique. By intraoral approach, we filled the defect area with autogenous grain bone covered by collagen membrane, then observed the repairing effect of autogenous grain bone induced by combined membrane through comparing the preoperative and postoperative numeral photos and imageology detection.Results:Part of animal experiment:Experiment one: Gross sample, X-ray examination and histology detectionGross sample and X-ray examination: In the experiment group, defect was clear2 weeks postoperative, a lamellar of osteotylus had formed on the side where had been filled with grain bone, and x-ray image showed low bone density and aniso-texture in defect area. However, the defect of control group was obvious and almost identical with the original defect. After 4 weeks of operation, in the experiment group, new bone filled in the defect area where was near the far end, the middle and the cheek-glossa side, the white line of cortical bone was unclear. At the same time, in the control group, there was also new bone around the defect area, but bone trabecula was unobvious. Tightly contacting with collagen membrane which was combined with bone growth stimulated factors, the new bone began to grow towards the center of the defect, but hadn't overlayed the mandibular defect completely. By comparison, there was great difference in the growth velocity between two groups. Comparing with the control group, in the experiment group, bone density was higher and new bone grew faster, but results of both groups were lower than the primitive bone. Although the defect area decreased more than that of 2 weeks postoperative, it was still obvious in the control group. Neogenetic bone of both sides possessed hard texture and shaggy surface at 8 weeks after operation. In the experiment group, X-ray displayed hyperosteogeny in defect area, with uniformly heightened density and irregular bone line, and cortical bone was continuous with a little lower density. Yet the defect area of the control lateral was much larger than that of the experimental lateral, and cortical bone was discontinuous in control group. 12 weeks after operation, in the experiment group, density of the new bone and the direction of trabecular pattern were close to the primitive bone, the collagen membrane combined with bone growth stimulated factors had been absorbed completely, and new osteotylus could be found at the defect area near the membrane. Bone density of area near the center of the defect was close to that of the peripheral area, and bone density of the region near the edge of the defect was almost coincident with that of the normal bone. As to the control group, the quantity of new bone was less than the experiment group, so did the bone density. There wasn't obvious bone growth in one sample of the control group, besides crude borderline. 24 weeks postoperatively, in the experiment group, density of new bone and the direction of trabecular pattern were coincident with the primitive bone at the area near the far end, middle and edge of the membrane. On the contrary, there was no distinct difference in the control group except that bone density near the center of the defect area of several samples was a little lower than that of the experiment group.HE dyeing and test under microscope: In experiment group, when 2 weeks passed, it showed that fibrous tissue filled up, interstitial cell hyperplasied, blood capillary grew into the defect area, osteoplast proliferated and lamellar osteotylus had formed, residual grain bone had been wrapped up with osteoclast surrounding; but foreign body reaction was slight, a small quantity of macrophages emerged, and new bone trabecula was cancellous. When 4 weeks passed, the collagen membrane combined with bone growth stimulated factors was light grey without obvious absorption, new bone could be seen under the membrane, especially in the gap between the defect and membrane, a great amount of trabecula with indiscriminate alignment emerged with hyperplastic collagen fibers inserting among them, and inflammatory reaction was obvious with lots of lymphocytes and plasmacytes infiltrating which was distinct in area close to the defect center. At 8th weeks, the arrangement of bone trabecular recuperated a lot and returned to woven bone which had been gradually transiting to lamellar bone, some medullary cavity communicated filled with plenty of myelocytes and adipocytes, without remnant grain bone or inflammatory reaction. After 12 weeks, residue of collagen membrane was scarcely visible, lamellar bone formed which consisted of thick sclerotin and bone lacuna and without grain bone remains. 24 weeks postoperatively, new bone had maturated and rebuilt as lamellar bone which had no difference with the reception bone bed. As for control group, the result of the 2th week was similar to that of the 4th week. There were a great deal of granulation and scar renovation by microscope observation, with muscle fiber inserting, cell hyalinization and serious inflammatory reaction. There was still conspicuous inflammatory reaction when 8 weeks passed, and new bone trabecula was less with separated distribution. Muscle tissues inserting into bone defect area could still be found at 12th week, and new bone was thin. There was no significant difference from the experiment group except the area near the center at the 24th week postoperatively. Experiment two: Observation of ultrastructureObservation by scanning electron microscope: 1. Experimental sample of 2 weeks: there was a great deal of new fiber tissues of low electron density in the defect area, and the collagen fibers arranged according to defect direction on the whole. The interface between the area of defect and normal was clear. There was massive calcified deposition displaying as high density punctiform, little clumping and floccule image in the base material. 2. Experimental sample of 4 weeks: In the new bone area, electron density and calcification degree of bone matrix increased, whereas fiber composition decreased noticeably, crystal deposition was thick and compact which exhibited short rod-shape and big clumping, the amount of osteoplast increased which had long prominence and connected netlikely each other. In addition, more erythrocyte could be found, the distance between the defect area narrowed gradually and was filled with porous new bone, but bone density between the new bone and the original bone was still different obviously. Fibrous connection began to form between the new bone and the normal bone, new porous woven bone occupied most space, new bone trabecula developed with the ultrastructure presenting 'honeycomb appearance'. 3. Experimental sample of 8 weeks: Calcification degree of bone matrix continued to increase, the defect area was filled with mature bone tissue, new bone tissue changed from cavernous crumbly structure to compact constitution, new lamellar bone's arrangement possessed some directivity and Haversian System, with small amount osteoblast lying in bone lacuna. Bone density of the defect area was similar to that of the original with indistinct border line, new bone and normal bone mixed together as synostosis, and marrow cavity communicated. When 24 weeks passed, there was no significant difference between the experiment group and the control group.Experiment three: Quantitative detection of ossification 1. Bone density detection and statistics analysis: All detection indexes were indicated by X±S, applying factorial analysis of variance of randomization design. The result indicated that difference between the experiment group and the control group was significant (F=30.835, P=0.000); so did the difference between different times (F=252.3775, P=0.000); there were interactions between groups and times (F=23.842, P=0.000). It indicated that autogenous grain bone transplantation guided by collagen membrane combined with BMP2 might promote ossification and will have a good result in repairing mandibular defect.2. Four weeks postoperatively, bone density of experiment group was 19.902±1.313, higher than that of control group which was 16.512±1.002, the data near the center of defect of the 12th week was 21.536±1.176 and 21.051±1.650 respectively while the figure of other area was 22.226±1.946 and 21.878±0.936 correspondingly, and the result of the 24th week was 24.275±1.633 and 24.429±1.597 respectively. According to pairing t-test analysis, the difference of 4th week between experiment group and control group was significant (P=0.000), so did the result near the center of defect of 12th week (P=0.028). However, the differences between other two groups (bone density of other area of 12th week and bone density of 24th week) had no significance and were not statistically significant with P=0.437 and P=0.711 respectively.3. We applied single factor analysis of variance for comparison between different weeks within each group, and all differences were significant (P=0.000). This manifested ossification way was different between different times.4. Comparing difference of bone density detection of 12th week between the area near the center and the other area. According to pairing t-test analysis, the difference between the two areas of the experiment group was not significant (t= 1.533, P=0.138); on the contrary, the difference of the control group was significant (t = 2.358, P=0.027). This indicated that new bone had been clearly mature at 12th week, and there had been new bone surrounding the center of defect which had obvious difference with other edge region. However, in the control group, no difference and statistical significance existed between area near the center and the other area, which showed that ossification way was alike.5. Morphous dyeing and slice testing under microscope of bone tissueMicroscopic examination of Masson-Goldner three color dyeing sections: Observed the sections by ordinary electron microscope after dyeing, the bone matrix was green, on the edge of which was the red unmineralized osteoid, bone cells located in the bone trabecula, with red-dyed cytoplasm and grey-blue-stained karyon. In the experiment group, bone matrix was abundant, bone trabecula distributed widely in good shape, with a lot of bone cells among them. In the control group, however, we could find a great deal of osteoid, sparsely-spreaded bone trabecula as well as less bone cells.Microscopic examination of Von Kossa dyeing sections: Observed the sections by the fluorescence microscope, the tetracycline fluorescence deposited on the bone surface was yellow, and the calcein was green. The marked mineralized bone matrix deposition between the two fluorescence strips displayed the rate of deposit and mineralization. According to the results of the semi-automatic digital image analytical tool, the deposition rate of the experiment group is higher than the blank group.Clinical application:Experiment four: The assistedly directive role of 3D spiral CT reconstruction for repairing clinical cranio-maxillo-facial bone defect.The patients with mandibular defect and congenital osteodysplasty were scanned 0.65 mm per layer by 64-lamella spiral CT, then the acquired datum were transformed into facies cranii 3D image by means of surface projected display on the online image workstation. By analyzing the 3D image, diagnosis and treatment plan could be made. And preoperative design and postoperative result were compared.Experiment five: Clinical application of repair for cranio-maxillo-facial bone defect with autogenous grain bone induced by membrane guided bone regeneration technique.All operations of 7 patients were successful. Without wound infection after operation, all incisions healed up at I stage, the symmetry was obviously better than that of preoperation, and facial outline was satisfactory. By 1 month to 6 months' follow-up, 3D CT showed bone healing, with a little bone absorption but without decalcification and necrosis. The defect area hardened compactly as uninjured side. Satisfactory result had been made in all patients.Conclusions:1. Reliable ossification results could be acquired and healing time could be shorten by repairing cranio-maxillo-facial defect with autogenous grain bone induced by membrane guided bone regeneration technique, according to ossification indexes detection. In our study, we applied BME-10X collagen membrane invented by engineering institute of Chinese Academy of Medical Science. Been combined with rhBMP-2, the membrane has histocompatibility, absorbability and histo-regeneration promoting ability. The main mechanism of guided tissue regeneration is maintaining the defect space by preventing soft tissue from growing into the defect area, which is beneficial to the activity of osteoblast and other repair cells. When bone heals, bone formation promoting factors produced by paracrine and endocrine secretion accumulate in the bone formation area, but it seems insufficient to use the membrane barrier only when the defect area is much larger and the capability of local bone formation is much weaker. Combining BMP-2 with biologic absorbable promoting bone formation membrane (collagen membrane) could both prevent the soft tissue from growing into and increase the concentration of bone formation promoting factors in local area, which could accelerate bone healing and make up the disadvantages of more rapid absorption speed of collagen membrane to some extent. When autogenous grain bone was reimplanted into the defect region, something occurred including bone absorption, blood vessel and the tissue around it growing into, new bone grew inwardly from broken ends and replaced them. The process belonged to typical 'creeping substitution'. During this process, the main mechanism of bone healing was bone conduction, bone induction and bone formation by the means of membrane-osteogenesis.2. Detection of bone histomorphomery parameters had verified that good results can be acquired by repairing cranio-maxillo-facial defect with autogenous grain bone induced by technique of membrane guided bone regeneration, which can quantitively and visually describe the dynamic changes of tiny morphology of new bone tissue.3. Being a reliable means of ossification test, preoperative and postoperative 3D CT re-establishment of mandibula was very important for direction of individualized recovery. It also provided beneficial imageology direction for clinical application of repairing cranio-maxillo-facial defect with autogenous grain bone induced by technique of membrane guided bone regeneration.4. The clinical application of repairing cranio-maxillo-facial defect with autogenous grain bone induced by technique of membrane guided bone regeneration showed that intraoral approach was convenient, the area of bone removal could be controlled more exactly by grinded osteotomy, and the ossification effect was dependable. Moreover, the facial outline was much better after operation, with the approximately identical symmetry.