中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (47): 7589-7596.doi: 10.3969/j.issn.2095-4344.2015.47.008
• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇 下一篇
以羟基丁酸酯与羟基已酸酯共聚物为支架材料构建组织工程喉形态软骨
孙安科1，孟庆延2，李万同2，刘松波3，陈 伟4
- 解放军沈阳军区总医院，1耳鼻咽喉科，2烧伤与整形美容科，3显微外科，4医学实验科，辽宁省沈阳市 110016
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收稿日期:2015-09-12出版日期:2015-11-19发布日期:2015-11-19 -
通讯作者:孙安科，解放军沈阳军区总医院耳鼻咽喉科，辽宁省沈阳市 110016 -
作者简介:孙安科，男，1962年生，河南省南阳市人，汉族，1985年解放军第二军医大学毕业，博士，主任医师，主要从事软骨组织工程与喉软骨功能重建研究。 -
基金资助:沈阳军区总医院院级重点基金课题(zy2009z0019)
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) as a scaffold to construct tissue-engineered larynx-shaped cartilage
Sun An-ke1, Meng Qing-yan2, Li Wan-tong2, Liu Song-bo3 , Chen Wei4
- 1 Department of Otolaryngology, General Hospital of Shenyang Military Area Command, Shenyang 110016, Liaoning Province, China
2 Department of Burns and Aesthetic Medicine, General Hospital of Shenyang Military Area Command, Shenyang 110016, Liaoning Province, China3 Department of Microsurgery, General Hospital of Shenyang Military Area Command, Shenyang 110016, Liaoning Province, China4 Department of Experimental Medicine, General Hospital of Shenyang Military Area Command, Shenyang 110016, Liaoning Province, China
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Received:2015-09-12Online:2015-11-19Published:2015-11-19 -
Contact:Sun An-ke, Department of Otolaryngology, General Hospital of Shenyang Military Area Command, Shenyang 110016, Liaoning Province, China -
About author:Sun An-ke, M.D., Chief physician, Department of Otolaryngology, General Hospital of Shenyang Military Area Command, Shenyang 110016, Liaoning Province, China -
Supported by:the Key Fund of General Hospital of Shenyang Military Area Command, No. zy2009z0019
摘要：
背景：再生预定形态组织工程软骨的研究为喉软骨病损的修复与重建提供了新思路与新方法。然而，由于喉软骨形态、部位与功能的特殊性，迄今在此领域软骨组织工程研究并未呈现出其应有的优势。
目的：探讨带蒂肌筋膜组织瓣构建组织工程喉支架形态软骨方法，为肌筋膜瓣复合组织工程化软骨修复重建喉软骨支架功能提供实验依据。
方法：采用溶剂浇铸、模压成形和颗粒滤沥方法制备喉支架形态聚羟基丁酸酯与聚羟基己酸酯共聚物聚羟基丁酸酯与聚羟基己酸酯共聚物聚羟基丁酸酯与聚羟基己酸酯共聚物生物材料塑形物，接种软骨细胞形成细胞-聚羟基丁酸酯与聚羟基己酸酯共聚物复合物，体外共同培养1周后用于体内植入。将新西兰白兔脊背部一侧骶棘肌及其筋膜制备肌筋膜组织瓣，采用筋膜衬里方法充填与包裹软骨细胞聚羟基丁酸酯与聚羟基己酸酯共聚物喉支架形态复合物，原位植入。将单纯聚羟基丁酸酯与聚羟基己酸酯共聚物喉支架体内植入的兔作为对照组。分别于术后6，12和18周取材，行大体形态观察、组织学和免疫组化检测评估喉支架形态组织工程化软骨成形与再生情况。
结果与结论：制备的聚羟基丁酸酯与聚羟基己酸酯共聚物多孔生物材料塑形物呈中空半面喇叭状，形似喉支架形态，乙醇静态容积测定孔隙率>90%。筋膜衬里的带蒂肌筋膜组织瓣血运丰富，可有效充填与包裹喉支架形态塑形物。不同时间点均获取形态维持良好的喉支架形态组织工程软骨，组织学和免疫组化检测均证实体内植入6周即可形成软骨组织，12周及18软骨组织进一步成熟，而对照组体内植入未检测到软骨组织。结果证实，带蒂肌筋膜组织瓣可保障血运，采用筋膜衬里的肌筋膜组织瓣充填与包裹方法可构建喉支架形态组织工程软骨。
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
引用本文
孙安科，孟庆延，李万同，刘松波，陈 伟. 以羟基丁酸酯与羟基已酸酯共聚物为支架材料构建组织工程喉形态软骨[J]. 中国组织工程研究, 2015, 19(47): 7589-7596.
Sun An-ke, Meng Qing-yan, Li Wan-tong, Liu Song-bo,Chen Wei. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) as a scaffold to construct tissue-engineered larynx-shaped cartilage[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(47): 7589-7596.
Quantitative analysis of experimental animals
Twelve male New Zealand white rabbits (nine in the experimental group and three in the control group) were observated at 6, 12 and 18 weeks after surgery, respectively. No animal died and no infection was found in the areas of implantation. Experimental data were statistically analyzed.
Establishment of PHBHH porous models
The porous PHBHH composite was prepared by solvent casting, compression molding and particulate filtering, and had a hollow, semi-trumpet shape, which was substantially similar to laryngeal cartilage morphology (Figure 1). Following the filtering and demineralization, the whole structure was porous and spongy. The porosity was (92±2)% measured using the ethanol static volumetric measurement method, and the pore size and thickness were found to be 100 150 μm and -1.5 mm, respectively.
Experimental observation results in vitro
At 24 hours after the inocu¬lation of chondrocytes into the hollow, semi-trumpet PHBHH biomaterials, the chondrocytes were observed to attach to the surface of the lower edge of the material under an inverted microscope. Following co culture of the chondrocytes and PHBHH for 1 week, a jelly like matrix, secreted by the chondrocytes, was visible at edge of the material (Figure 2A).
Scanning electron microscopy results
The simple PHBHH material was porous and spongy, exhibiting a pore size of 100-150 μm (Figure 2B). Scanning electron microscope showed that the cells (single, string or cluster) were densely distributed on the surface and in the cavernous, spongy pores of chondrocyte PHBHH complex. Mucus like stromal substances around the cells exhibited interlinking adhesions, and numerous small projections were visible under high magnification microscopy, which may have been fused matrix components secreted by the chondrocytes (Figure 2C).
General morphology
The materials were harvested at 6 weeks after implantation, and the myofascial tissues covering the implants and connective tissues were wrapped together. Dense and tiny blood vessels were distributed in the connective tissues. Subsequent to stripping away a part of the wrapped tissues, it was observed that the filled myofascial tissue was closely in contact with the implants. After the steel wire support frame, filled packages and fascia tissues were removed, it was found that the general form of the implants in the two cases was consistent with the morphology prior to implantation (Figure 3A). The specimens possessed certain hardness and braced force with smooth inner and outer surfaces. In one case, the implant collapsed, showing angular release and a loss of the predetermined hollow, semi-trumpet shape (Figure 3B). At 12 weeks, only two complete specimens were harvested from the three experimental implants, and the general form was similar to a hollow, semi-trumpet, tissue engineered laryngeal cartilage. When harvested at 18 weeks, the implant shape of the three cases was consistent, and the milky white, hollow, semi-trumpet, tissue engineered laryngeal cartilage exhibited a realistic shape (Figure 4). In the control group, one implant was removed at each corresponding time point. The retained hollow, semi-trumpet shape of the gross specimens (Figure 3C) in the control group at 6 weeks was significantly less than that of the experimental group (Figure 3A) at the same time point. At 12 and 18 weeks after implantation, the gross morphology of the implants in the control group almost disappeared, and only the built in medical steel support frame was visible in the implant area.
Hematoxylin-eosin staining results
At 6 weeks after implantation, the cells of the experimental group exhibited a circular, oval or polygonal morphology, and single cells were scattered. “Impurities”, resulting from the incomplete degradation of PHBHH, were visible among the cells, and inflammatory cell infiltration was present around the cartilage (Figure 4A). At 12 weeks, the majority of the cells exhibited an oval morphology; 2-3 cells were commonly clustered together and showed a uniform stromal staining. The morphology of the cells at 18 weeks was similar to that at 12 weeks; the “impurities” disappeared, but a small number of inflammatory cells remained in the surroundings. In the control group, cartilage like cells were not visible, the collapse of the pores and degradation of the material coexisted at 6 weeks, and few other tissue cells were scattered on the material surface (Figure 4B).
Masson’s trichrome staining results
Pale green objects were observed in the chondrocytes and cartilage matrix following cytoplasmic staining of the experimental group at the 6th week, and the presence of the green pollutants increased significantly at the 12th week, with deeper staining (Figure 5A). The cell morphology, distribution and coloring at 18 weeks were not different from the observations at 12 weeks.
Alcian blue/periodic acid Schiff reaction staining results
The matrix was pale purple at 6 weeks, and purple at 12 and 18 weeks after implantation (Figure 5B).
Immunohistochemistry for type II collagen results
At 6 weeks after implantation, immunohistochemistry for type II collagen in the experimental group (Boster Biotechnology Co., Ltd., Wuhan, China) revealed weakly positive staining, which was mainly distributed in the cytoplasm, with very small amounts in the matrix. At 12 weeks, the positive staining in the cytoplasm was significantly enhanced, and increased type II collagen positive staining was detected in the matrix (Figure 5C). Similar results were observed at 18 weeks.
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Tissue engineering technology provides new ideas and techniques for repair and reconstruction of laryngeal cartilage erosion and has obtained many achievements[1-3]; however, due to the special morphology, location and function of laryngeal cartilage, tissue engineering research has not, to date, exhibited its full advantages in the reconstruction of laryngeal cartilage[4]. This is not only due to the large quantity of seed cells and biomaterials required for the construction and shaping of the tissue engineered laryngeal cartilage, but also due to the difficulty in the construction of ideal irregular, hollow, three dimensional structure. To construct the tissue engineered laryngeal cartilage with a hollow and semi trumpet shape, the biomaterials should show good biocompatibility, biodegradability and structures inside the pores, as well as have sufficient mechanical strength and flexibility[5]. How to ensure that the tissue-engineered cartilage maintains its hollow and semi trumpet shape and structure and how to develop the potential of cartilage reshaping applications are two problems currently faced in the formation of tissue engineered, hollow, semi-trumpet laryngeal
cartilage. To explore hollow structural biomaterials that are more suitable for shaping and tissue engineered cartilage regeneration is the only way to improve laryngeal cartilage repair and reconstruction by cartilage tissue engineering. In the present study, on the basis of previously published tissue engineering findings[6-8], the new biomaterial poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHH) was used in the shaping of tissue engineered laryngeal cartilage with a hollow and semi-trumpet shape, and then its compatibility of with chondrocytes was investigated. The aim of the study was to determine the feasibility of building tissue-engineered laryngeal cartilage with a hollow, semi- trumpet shape that is filled and encapsulated using pedicled myofascial flap, in order to provide an experimental basis for construction of tissue-engineered laryngeal cartilage with a hollow, semi-trumpet shape through the transplantation of myofascial tissue[9].
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
MATERIALS AND METHODS
Design
A study about biomaterial modeling and cartilage regeneration.
Time and setting
From October 2011 to April 2013, the study was carried out in the Center of Labaratory Animals and Experimental Medicine, General Hospital of Shenyang Military Area Command, China.
Animals
Twelve male New Zealand white rabbits (age, 5-6 months; weight, (2.5±0.5) kg; license no. 20060828001) supported by the Center of Laboratory Animals and Experimental Medicine, General Hospital of Shenyang Military Area Command were enrolled in this study. This experiment was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the General Hospital of Shenyang Military Area Command.
Biomaterials
PHBHH flocculent material (molecular weight, 600 000) was provided by the Department of Chemical Engineering, Tsinghua University, China.
Methods
Chondrocyte culture
Costal and articular cartilages were obtained under sterile conditions from young New Zealand rabbits of both genders, aged 3-7 days. The cartilage specimens were cleaned, washed twice with 0.1 mol/L PBS containing 200 U/mL penicillin and streptomycin, and digested with 0.25% trypsin for 2 minutes. Following digestion, the cartilage specimens were washed with PBS three times, cut into pieces (1.0-2.0 mm3) and washed again with PBS. The pieces were then placed in a small 50 mL beaker. Type II collagenase (0.3%; Sigma Aldrich) was added and stirred
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Model preparation
Shaping was performed in reference to the full morphology of adult laryngeal cartilage, and the plastic models were prepared by 3:1 shrinking. Polytetrafluoroethylene was taken as the mold material, engraved the form of the corresponding female die, which was comprised by an inner core and two outer plates.
Poration and shaping of the PHBHH biological material
The biological materials exhibiting a hollow, semi-trumpet laryngeal cartilage like morphology were prepared by solvent casting, compression molding and particulate filter leaching methods[10]. In brief, quantitative PHBHH flocculent material (molecular weight=600 000) was placed inside a spherical, heat resistant glass container, and a certain percentage of chloroform solvent was added. The mixture was then heated for reflux under sealed conditions, stirred with a magnetic stirrer and rapidly poured into a wide neck flask containing sodium salt (screening of 150-200 μm). Once the solution formed a uniform thin paste, the bottle was sealed and placed at room temperature overnight (making the material naturally penetrate the salt layer). The mixture of the material and the salt was firstly prepared into a sheet, then wrapped around the mold core portion, closed around the outer mold and fixed by metal fixture; the liquid was then compressed into the mold. The samples were placed in the fume cupboard for ≥ 48 hours for full evaporation of the chloroform and subsequently placed in a vacuum pump suction filter for 12 hours to remove any possible residual chloroform. The obtained samples were set within an inner metal mesh, suspended in glass containers containing distilled water, magnetically stirred and filtered for desalination. The triple distilled water was replaced at least three times. The desalinated samples were dried naturally for porosity determination.
Determination of PHBHH porosity
The liquid displacement method[11] was used in the determination of PHBHH porosity, Due to the lipophilic nature of the PHBHH, ethanol was used instead of water. This facilitated the penetration of the liquid into the material. The ethanol was added with a volume scale (V1) into a sealable test tube and the test sample (mass, M) was weighed and then also added to the tube. The tubes were subsequently sealed, opened after 8 hours (to release the gas) and closed again. After 24 hours, the ethanol penetrated sufficiently into the pores within the sample, and the volume was recorded as V2. Subsequent to removing the sample saturated with ethanol, the volume of ethanol in the tube was recorded as V3. The material density was calculated using the following formula: ρ=M/(V2-V3). The porosity (%) was calculated using the following formula e=(V1-V3)/(V2-V3)×100.
Assistance for shaping and adhesion
The shaping of the hollow, semi trumpet PHBHH objects was aided by the inner surface of a steel wire with a diameter of 0.4 mm, which was uninterruptedly woven into the form of two approximate rings and three longitudinal supports. Compared with the PHBHH with the hollow semi-trumpet shape, the larger ring fluctuated correspondingly up and down along the shape of the corners and edges of the scaffold, and mainly helped the shaping of the edges and rear parts with less material of the PHBHH with hollow semi trumpet shape. The smaller ring was nearly round, which assisted in the shaping of the small end of the hollow, semi trumpet PHBHH, and the three longitudinal wire supports were located in the sides of the PHBHH shape and in the middle of the front. The steel support frame was placed tightly inside the PHBHH model with no special fixation. The hollow, semi trumpet PHBHH constructions and the steel wire support frame were disinfected by immersion into 75% alcohol and then rinsed three times with PBS, prior to being dried under sterile conditions. Adhesion was promoted by immersing the PHBHH and the frame into sterile poly L-lysine aid (relative molecular mass, 1.89×107) for 1 hour, prior to drying.
Compounding of chondrocytes with PHBHH and culturing in vitro
Following the adjustment of the cell suspension to a density of 5×1010/L, the chondrocytes were inoculated in the pre dried shaping materials by culture medium containing about 30% of wet, a small amount of cell suspension each time when inoculated, was scattered and multicasted to make the cells distribute as even as possible[12]. The inoculated cells of the inner surface of the shaping hollow material were pipetted by bend head. The cells were placed in a CO2 saturated humidity incubator at 37 ℃ and 5% CO2 for 0.5 hour, and then removed to adjust the up and down placing state of the shaping material. The cell suspension around the bottom of the material was sucked out and inoculation was performed from top to bottom twice, prior to further incubation for 1 hour. Fresh culture medium was then added slowly along the sidewall so that the composite was infiltrated by 2.0-3.0 cm. The medium was changed once every 48 hours. Cell growth and adhesion in the composite edge were observed under an inverted microscope. The shaped material loaded with chondrocytes was used in the experimental group, while a control group was established by shaping the PHBHH without the chondrocytes. All other steps in the two groups were identical. Each 5 mm×5 mm sheet like chondrocyte PHBHH composite was fixed with 2.5% glutaraldehyde, dehydrated progressively with alcohol, dried, mounted and observed using light emitting plasma spraying with scanning electron microscopy.
Grouping
Twelve male New Zealand white rabbits were randomly divided into an experimental group (n=9) and a control group (n=3). Three rabbits in the experimental group and one rabbit in the control group were observated at one time point (totally three time points: 6, 12 and 18 weeks after surgery).
Design of the pedicled myofascial flap
Intravenous anesthesia was induced with 3% barbiturate. The side of the rabbit back skin was disinfected and shaved, and the middle portion of the sacral spine muscle and fascia was isolated from the distal side of the rabbit’s back to form the myofascial tissue flap with the pedicle for the proximal and free muscular fasciae for the distal. The filled myofascial flap with a conical hollow portion was designed according to the size of the chondrocyte PHBHH composite. The fascial layer of myofascial flap was then stabbed into the form of a net with a scalpel blade for the cone of filling the myofascial flap in the prepared hollow part. During flap preparation, the sacral spinal fascia in the sides of the depression was freed, simply sutured, connected and placed in the recessed slots, and the fascia in the depressed trench was subjected to poration for the in situ implantation of the PHBHH chondrocyte complex. Following implantation, the remaining sacral spine muscle and fascia were separated to enclose the chondrocyte PHBHH complex. Autologous fascia tissues were used for each implanted chondrocyte PHBHH composite.
In vivo implantation
The chondrocyte PHBHH composites were co cultured in vitro for 7-10 days, until abundant extracellular matrix was observed using inverted microscopy. The composites were removed subsequent to reaching a certain degree of support (hardness). The in situ implantation was designed, filled and wrapped in the body according to the design of the myofascial tissue, and the distal portion of the filled myofascial flap was fixed. The subcutaneous skin was sutured. Each batch of surgical procedures was performed by the same group of technicians, and the surgical procedures and technology used were consistent. The rabbits were administered an intramuscular injection of 800 000 units penicillin, and the surgery was then repeated the next day for a total of four times.
Evaluation of poration, shaping and cellular compatibility of PHBHH
The PHBHH composites were observed and assessed for shapes similar to the hollow, semi-trumpet laryngeal cartilage morphology. The composite pore continuity and chondrocyte adhesion, distribution and growth were observed using scanning electron microscopy.
Construction of the tissue engineered larynx-shape cartilage
The animals were anesthetized and the implants were removed: Three rabbits from the experimental group and one from the control group were selected at each time point (6, 12 and 18 weeks after surgery), respectively. The gross morphology of the tissue engineered laryngeal cartilage was observed, and hematoxylin-eosin staining, Masson’s trichrome and alcian blue/periodic acid Schiff reaction (AB/PAS) staining were performed, as well as collagen type II immunohistochemical detection for the evaluation of cartilage formation.
Main outcome measures
Construction of PHBHH porous models, chondrocyte PHBHH complex, general morphology and histological observation of tissue engineered cartilage were measured.
Statistical analyses
All data was statistically analyzed with SPSS 11.0 software. The measurement data were represented as mean±SD. The comparison between groups that met the normal distribution was done using independent sample t-test and one-way analysis of variance, and the comparison that did not meet the normal distribution was done using the nonparametric test. A value of P < 0.05 was considered statistically significant.
Different from the regeneration of sheet cartilage tissue by tissue engineering technology, the construction of hollow, semi-trumpet, tissue engineered laryngeal cartilage has a higher demand for biocompatibility, biodegradability, pore structure, mechanical strength and the shaping of extracellular matrix materials[13]. Biomaterials that are currently used in cartilage tissue engineering include natural biomaterials, such as collagen, fibrin and chitosan; synthetic biomaterials, such as polyglycolic acid; microbial biosynthetic material, such as types of biological polyhydroxyalkanoates and copolymers; and composite materials, such as polylactic acid collagen and fibrin polyurethane[14-19]. The biomaterials each have individual advantages and disadvantages in terms of biocompatibility, degradation suitability, cell adhesion, metabolic microenvironment, mechanical strength, shaping and flexibility. Natural biomaterials lack sufficient mechanical strength, while artificial and compound biomaterials are expensive and involve complex processing, but are easy to shape.
This study has raised the potential for a common transfer transplantation with complex of the tissue flap and hollow, semi-trumpet, tissue engineered cartilage, and has indicated the success of the myofascial flap filling and wrapping method in generating hollow, semi-trumpet, tissue engineered laryngeal cartilage for functional repair and reconstruction. The study has also demonstrated the potential of the mucosal epithelial transformation of an ideal hollow cartilage surface; however, the results of this study are preliminary, the design of laryngeal cartilage scaffolds based on this design concept as well as the construction, transplantation and practical applications of the composite tissues should be the direction of future studies.
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
Polyhydroxyalkanoate is a natural substance produced by microorganisms and exhibits characteristics such as good biocompatibility, no induction of inflammation, no degradation and ease of rejection[20]. As more types of polymers become available, and as the compatibility among the polymers is enhanced, tissue engineering scaffolds with improved strength, toughness, biocompatibility and controlled degradation are likely to be obtained through the advantages resulting from the combination of different polymers.
PHBHH, a copolymer of poly-3-hydroxybutyrate and
poly-3-hydroxyhexanoate ester, not only has the advantages common to biological materials belonging to the polyhydroxyalkanoate class, but also can be found at low prices and from abundant sources. In this study, PHBHH was made into a hollow, semi-trumpet, tissue engineered, larynx shape model by solvent casting and compression molding methods; the material maintained a good shape subsequent to desalting by filtering and exhibited considerable mechanical strength. The material was porous and spongy under the electron microscope, the pore size was consistent with the screening of the salt, the porosity was high, which was in line with the basic requirements for a porous structure, and the model showed a certain degree of mechanical strength. Following the co culture of the cartilage cells with the PHBHH composite material in vitro, the model showed good cell adhesion, distribution and vigorous growth; however, the mechanical strength was still insufficient to maintain the predetermined shape upon implantation, which highlighted the necessity of the auxiliary, in built support frame. The implantation of simple PHBHH laryngeal cartilage scaffolds, without cells, was taken as the control group; after 6 weeks an increased loss of general shape was observed in the control group compared with the experimental group, in which a chondrocyte PHBHH composite had been implanted. Under the microscope, no cartilage like structure was observed in the control group, and more incompletely degraded impurities of PHBHH were apparent in the cartilage tissue. The shape of the control group implants was lost at the 12th and 18th weeks, suggesting that implantation of the material for 6 weeks resulted in significant, but not complete, degradation and absorption. Implantation of PHBHH alone, without seed cells, led to an ineffective generation of cartilage tissue. By contrast, after 12 weeks of in vivo implantation in the experimental group, the mature, hollow, semi-trumpet, tissue engineered laryngeal cartilage could be obtained.
Although basic research in cartilage tissue engineering has achieved encouraging results, the functional repair andreconstruction of tissue engineered laryngeal cartilage has not yet been implemented in a practical sense. The factors impeding this implementation are the blood supply and mucosal and supportive defects of the regenerated cartilage. The limitations in the regeneration of cartilage could be overcome by combining the advantages of tissue engineering technology and the predetermination of cartilage morphology with the advantages of the use of a tissue flap to provide a blood supply. Through this strategy, and using the characteristics of epithelization, an affiliation could be achieved between the fibrous tissues and the tissue flap. The tissue flap could be prepared using the design principle of filling the lining of the cavity fascia and wrapping the outer cavity with muscle to adhere to the tissue engineered cartilage. The complex could then be used to reconstruct the laryngeal cartilage with scaffold functions according to the common nearest transfer or graft method. This technique is likely to overcome the problems in vascularization, mucosalization and support in cartilage repair and reconstruction for laryngeal cartilage.
Based on the above idea, the present study attempted to take a fascia tissue lining and use myofascial tissue flap filling and wrapping technology to build a predetermined, hollow, semi-trumpet, tissue engineered laryngeal cartilage. In the experimental procedure, the myofascial tissue of the fascial portion was placed on the surface of the proposed cartilage construction for contact. Fascia poration not only enhanced blood circulation, but was also conducive to adhesion between the structures. Experimentally designed muscle flap was filled with a rich blood supply, while the flap off recessed slots also formed equally rich blood supply. The cultured chondrocyte hollow, semi-trumpet PHBHH composite was placed off the depression of the tissue flap and cultured for a certain period of in vitro. The cavity was filled with fascia and muscle lining, and the periphery was supported by the tissue around recessed slots. The upper part of the complex was covered and wrapped with myofascial tissues. The results of the study showed that the application of myofascial tissue with a flap blood supply with functional filling and wrapping technology could successfully lead to the generation of hollow, semi-trumpet, tissue engineered laryngeal cartilage. Difficulties may arise, however, in avoiding the collapse of the samples, considering the multiple steps in the construction of the hollow, semi-trumpet, tissue engineered laryngeal cartilage.
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
实验将新型生物材料聚羟基丁酸酯与聚羟基己酸酯共聚物塑形成中空半喇叭形态，并与软骨种子细胞相复合，采用筋膜衬里、肌筋膜瓣充填与包裹技术构建中空半喇叭形态组织工程喉软骨，为肌筋膜瓣复合组织工程化软骨修复重建喉软骨支架功能提供实验依据。 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
聚羟基烷酸酯类材料的特点？是由微生物产生的聚酯类天然物质，具有生物相容性好、无致炎性、无排斥性和易降解等特点。 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
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