新型介稳定β－钛合金在喉、气管缺损重建中的应用与研究
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摘要
各种原因导致的喉、气管缺损是耳鼻咽喉科的常见病与多发病,对于喉气管缺损的修复与重建目前仍无标准的治疗。由于喉气管缺损的原因与严重程度不同给临床治疗带来了极大的困难。多年来研究喉气管缺损的修复与重建以关注假体材料的选择、加工与重建方式为主,近年来一些学者亦通过动物模型研究各种修复材料与长段气管缺损的重建方法,发现假体的血管化与上皮化仍是我们目前临床面临的挑战。然而,新型介稳定β-钛合金材料对于喉、气管缺损的修复与重建目前尚无报道。
目的:观察并探讨新型介稳定β-钛合金(TLM)材料在犬的喉、气管缺损修复与重建中的有效性,为喉、气管缺损的治疗提供一种可选择的生物材料,同时为临床治疗提供新的理论依据。
方法:1.采用激光切割方法,将钛合金板制成微多孔结构。高压消毒后,置于6孔板中,将小鼠NIH-3T3单细胞悬液按1×105细胞/毫升的密度加入材料表面培养,四小时后取一片材料做扫描电镜观察。测量孔径大小并做图像分析,计算孔隙率。并对材料表面的细胞活力与形态进行观察。
2.取5只犬,将犬的喉与气管充分暴露,切除环状软骨及第1气管软骨环的前1/3形成喉前裂开,将“◇”形的生物材料替代切除的部分喉气管组织,按计划术后3至6个月对受试动物进行气管镜检测。3.采用多孔TLM合金作为10只杂种犬的前喉气管组织再生支架。修复体长度为20mm,修复与重建前环状软骨及第1气管环的缺损,进一步评估TLM合金在该治疗中的有效性。4.取20只杂种犬,随机分成2组:TLM合金组与Ti-2组,每组各10只。切除环状软骨及第1、2气管软骨环,将两种“C”形的管状生物材料替代切除的部分喉气管缺损,按计划术后1、4及12周对受试动物分期进行喉气管镜检查、CT扫描及组织病理学检测。5.选择5只杂种犬行颈部切除5厘米长段气管,将多孔TLM制作成人气管形状以修复环周气管缺损。按计划植入3至8个月后处死动物行内镜检查、CT扫描和病理分析。
结果:1. TLM合金表面凹凸不平,呈多孔结构,孔径为70～90μm,孔隙率为32%。细胞表现出有足突和伪足的正常细胞形状。2.术后在所有受试犬中均未见到任何的气道阻塞。未见修复体的移位暴露,内表面无肉芽组织生长及吻合口裂开。3.一只犬于术后一周复查因麻醉意外而死亡;一只犬于术后约一个月因肺部感染而死亡;剩下的8只犬均于术后3至8个月处死;处死时发现所有修复体与周围宿主组织融合。术后内镜检查显示:无1只发生气道狭窄,其中4只长有肉芽组织,一只发现多孔TLM合金板暴露,未观察到吻合口的裂开。然而,所有这些犬均无临床症状。光镜与电镜结果显示:在假体内腔表面有一个正常的粘膜组织生长,及在重建的喉气管内腔位置未观察到任何的可见肉芽组织。4.术后在两组中均未出现吻合口漏。两种材料的修复体均表现出了良好的生物机械强度。组织学检测结果显示:TLM合金修复体周围的成纤维细胞在早期就从网孔长入,且修复体内腔吻合口部位可见有丰富的纤毛柱状上皮,纤毛分布密度也均匀,而在Ti-2组中,修复体周围组织长入较慢,且上皮分布明显不均。5.该假体的机械性能好,假体与周围组织结合紧密,结果充分展示了假体的生物相容性与耐腐蚀性,且没有产生任何的气管狭窄与空气的渗漏,并允许少量的肉芽组织正常长入气管内腔。
结论:1.首次采用TLM合金材料应用于喉、气管缺损的修复与重建。TLM合金材料具有良好的生物相容性、耐腐蚀性及机械性能,特别是其弹性模量(30～50Gpa)最接近骨与软骨,为喉气管缺损的重建提供了良好的应力分布。2.首次采用激光切割方法,将钛合金板制成微多直孔结构的喉气管假体,其微孔大小定位在70-90um,孔隙率为30%～35%,厚度为0.5mm。假体植入后发现在早期阶段因周围结缔组织从网孔的长入而保证了气密性,且其微血管的形成为假体腔内的快速上皮化提供了条件,从而最终克服了假体内腔的狭窄。同时发现本重建过程比目前所有试验的其它多孔材料要快。我们进一步证明上皮化结果与气管两端及假体孔隙进入的含丰富毛细血管网的结缔组织有关。3.TLM合金是一种很有潜力的新型喉气管缺损的修复材料,对未来临床的应用具有进一步深入研究的意义与价值。
Larynx and tracheal defect caused by a variety of reasons is a common and frequently-occurring disease in otolaryngology. There is no standard treatment in numerous reconstruction procedures at present. Different reasons and varying degress of the laryngotracheal defect bring about huge difficult for clinical therapy. Researchs of repair and reconstruction of laryngotracheal defect have paid mainly attention to option and process,as well as to pattern of reconstruction for a long time. Recently some researchers have studied various of repair material and reconstruction method of longer tracheal defect through animal model,and have found that vascularization and epithelialization of the prosthesis remains a challenge problem for the otolaryngologist. However, There are still no reports on the novel meta-stableβ-type titanium (TLM )alloy prosthesis for replacement of larynx and tracheal defect.
Objective: To explore and evaluate the efficacy of the larynx and tracheal reconstruction via in TLM in a canine model, to provide a selectable material for the treatment of laryngotracheal defect. And to provide evidences for the clinical therapy for the reconstruction method of laryngotracheal defect.
Methods : 1. It was made by cutting a TLM2 alloy plate and drilling tiny holes with laser on it. Rat NIH-3T3 cells were plated at a density of 1×105cells/ml on porous TLM samples placed in 6-well plastic plates after high pressure sterilization. Then they were cultured for 4 hours .The material was scaned by an electron microscope. The microscopic photos were analyzed by image pattern analysis to measure the porous size and porous rate.The cells on the samples surface were observed to assess its activit and shape. 2. Porous TLM alloy was used as a tissue scaffold for airway regeneration in five dogs. the larynx and the trachea were exposed. The anterior 1/3 part of the cricoid cartilage (cover the cricothyroid membrane) and 1 cervical trachea were simultaneously resected by cold instruments form an artificial anterior larynx open. The“◇”scaffold material was implanted into the resultant defect. Endoscopic examination was undertaken periodically for a postoperative period of 3 to 6 months in all dogs.3. A porous type of cricotracheal prosthesis made of porous TLM alloy is designed. This prosthesis (20 mm in length) is used to reconstruct the cricoid cartilage and the first tracheal ring in 10 mongrel dogs and its efficacy is evaluated. 4. Twenty adult mongrel dogs were chosen , divided randomly two groups. The TLM prosthesis were implanted into one group of dogs(n=10),and the pure titanium(Ti-2) prosthesis were implanted into another group of dogs(n=10). Cricoid cartilage and the first 1,2 tracheal ring reconstruction was performed with the biomedical materials both the TLM and the Ti-2 prosthesis (25 mm in length) in dogs. All dogs were euthanized in 1, 4 and 12 weeks and tracheal CT scan, tracheal endoscopy,histopathological examination and scanning electron microscopic examinations were performed. 5. 5 cm long segment of the cervical trachea was resected in 5 dogs. Porous TLM is fabricated into the shape of a human trachea to repair circumferential tracheal defects. The planned period for the implantation was from3 to 8 months before the sacrifice of the animals for endoscopic examination, CT scan and histological analysis.
Results: 1. Scanning electron microscopic photos showed that the surface structure of the samples was porous and the diameters varied from 70 to 90μm.The image analysis demonstrated that the porous rate was 32%. Cells appeared to have a normal cellular shape with lamellipodia and pseudopodia. 2. Endoscopic examination showed the implant located perfectly and the airway is wide enough for breath. There was no airway stenosis in any of the dogs. Granulation was observed in no cases, and anastomotic dehiscence was not observed. 3. One dog died due to an accident with anesthesia at 1 week, one dog died of pneumonia about 1 month after operation,and six dogs were killed between 3 and 8 months; At sacrifice, all the prostheses had become completely incorporated into the host tissue. Endoscopic examination showed no airway obstruction for a postoperative period of 3 to 8 months in 8 dogs. Granulation was observed in 4 cases, and the porous TLM alloy plate was exposed in 1 case, but these dogs were asymptomatic. Light microscopy and electron microscopy showed a normal mucous membrane growth on the prosthesis surface and no visible granulation tissue in the reconstructed cricoid and tracheal cavity. 4. Two type of the biomedical materials showed excellent mechanical strength after implantation. the Porous TLM alloy prothesis was present air-tightness after implantation at early period. The CT scan,endoscopic examination showed no apparent airway obstruction for a postoperative period of 1 to 12 weeks in TLM group,only 3 cases was free of airway obstruction in Ti-2 group. Histologic result showed a normal mucous membrane with normal cilial growth on the TLM implant.5. The mechanical performance of the prostheses was good and the protheses were integrated by the surrounding tissue.Results showed an adequate consistency of prosthesis, adequate tolerance without producing tracheal stenoses, and impermeability to air, allowing a correct invasion by granulation tissue.
Conclusion : 1. For the first time, we conclude that this TLM alloy is a kind of excellent biomedical material in replacement of larynx and tracheal defect due to its traits as having moderate strength,toughness matching,low elastic modulus(30～50Gpa),corrosion resistance,which provide a good stress distribution for reconstruction of laryngotracheal defect. 2. For the first time, It was made by cutting a TLM alloy plate and drilling tiny holes with laser on it. The diameter of the porosities was 70~90μm,giving a porosity of 30~35%, thickness was 0.5mm.We found that good incorporation of the prosthesis pore into the host tissue to maintain the air-tightness of the prosthesis after implantation in the early period,and confluent regeneration of the epithelium over the TLM alloy to overcome stenosis of prosthesis lumen. This process was faster than any found in any other porous tracheal prosthesis so far tested. We further proved that epithelialization results from capillary invasion through the prosthetic pores and growth from both tracheal ends.3. The TLM alloy is a potential new biomaterial for laryngotracheal defect reconstruction,which has highly research value for clinical application in the future.
目的:观察并探讨新型介稳定β-钛合金(TLM)材料在犬的喉、气管缺损修复与重建中的有效性,为喉、气管缺损的治疗提供一种可选择的生物材料,同时为临床治疗提供新的理论依据。
方法:1.采用激光切割方法,将钛合金板制成微多孔结构。高压消毒后,置于6孔板中,将小鼠NIH-3T3单细胞悬液按1×105细胞/毫升的密度加入材料表面培养,四小时后取一片材料做扫描电镜观察。测量孔径大小并做图像分析,计算孔隙率。并对材料表面的细胞活力与形态进行观察。
2.取5只犬,将犬的喉与气管充分暴露,切除环状软骨及第1气管软骨环的前1/3形成喉前裂开,将“◇”形的生物材料替代切除的部分喉气管组织,按计划术后3至6个月对受试动物进行气管镜检测。3.采用多孔TLM合金作为10只杂种犬的前喉气管组织再生支架。修复体长度为20mm,修复与重建前环状软骨及第1气管环的缺损,进一步评估TLM合金在该治疗中的有效性。4.取20只杂种犬,随机分成2组:TLM合金组与Ti-2组,每组各10只。切除环状软骨及第1、2气管软骨环,将两种“C”形的管状生物材料替代切除的部分喉气管缺损,按计划术后1、4及12周对受试动物分期进行喉气管镜检查、CT扫描及组织病理学检测。5.选择5只杂种犬行颈部切除5厘米长段气管,将多孔TLM制作成人气管形状以修复环周气管缺损。按计划植入3至8个月后处死动物行内镜检查、CT扫描和病理分析。
结果:1. TLM合金表面凹凸不平,呈多孔结构,孔径为70～90μm,孔隙率为32%。细胞表现出有足突和伪足的正常细胞形状。2.术后在所有受试犬中均未见到任何的气道阻塞。未见修复体的移位暴露,内表面无肉芽组织生长及吻合口裂开。3.一只犬于术后一周复查因麻醉意外而死亡;一只犬于术后约一个月因肺部感染而死亡;剩下的8只犬均于术后3至8个月处死;处死时发现所有修复体与周围宿主组织融合。术后内镜检查显示:无1只发生气道狭窄,其中4只长有肉芽组织,一只发现多孔TLM合金板暴露,未观察到吻合口的裂开。然而,所有这些犬均无临床症状。光镜与电镜结果显示:在假体内腔表面有一个正常的粘膜组织生长,及在重建的喉气管内腔位置未观察到任何的可见肉芽组织。4.术后在两组中均未出现吻合口漏。两种材料的修复体均表现出了良好的生物机械强度。组织学检测结果显示:TLM合金修复体周围的成纤维细胞在早期就从网孔长入,且修复体内腔吻合口部位可见有丰富的纤毛柱状上皮,纤毛分布密度也均匀,而在Ti-2组中,修复体周围组织长入较慢,且上皮分布明显不均。5.该假体的机械性能好,假体与周围组织结合紧密,结果充分展示了假体的生物相容性与耐腐蚀性,且没有产生任何的气管狭窄与空气的渗漏,并允许少量的肉芽组织正常长入气管内腔。
结论:1.首次采用TLM合金材料应用于喉、气管缺损的修复与重建。TLM合金材料具有良好的生物相容性、耐腐蚀性及机械性能,特别是其弹性模量(30～50Gpa)最接近骨与软骨,为喉气管缺损的重建提供了良好的应力分布。2.首次采用激光切割方法,将钛合金板制成微多直孔结构的喉气管假体,其微孔大小定位在70-90um,孔隙率为30%～35%,厚度为0.5mm。假体植入后发现在早期阶段因周围结缔组织从网孔的长入而保证了气密性,且其微血管的形成为假体腔内的快速上皮化提供了条件,从而最终克服了假体内腔的狭窄。同时发现本重建过程比目前所有试验的其它多孔材料要快。我们进一步证明上皮化结果与气管两端及假体孔隙进入的含丰富毛细血管网的结缔组织有关。3.TLM合金是一种很有潜力的新型喉气管缺损的修复材料,对未来临床的应用具有进一步深入研究的意义与价值。
Larynx and tracheal defect caused by a variety of reasons is a common and frequently-occurring disease in otolaryngology. There is no standard treatment in numerous reconstruction procedures at present. Different reasons and varying degress of the laryngotracheal defect bring about huge difficult for clinical therapy. Researchs of repair and reconstruction of laryngotracheal defect have paid mainly attention to option and process,as well as to pattern of reconstruction for a long time. Recently some researchers have studied various of repair material and reconstruction method of longer tracheal defect through animal model,and have found that vascularization and epithelialization of the prosthesis remains a challenge problem for the otolaryngologist. However, There are still no reports on the novel meta-stableβ-type titanium (TLM )alloy prosthesis for replacement of larynx and tracheal defect.
Objective: To explore and evaluate the efficacy of the larynx and tracheal reconstruction via in TLM in a canine model, to provide a selectable material for the treatment of laryngotracheal defect. And to provide evidences for the clinical therapy for the reconstruction method of laryngotracheal defect.
Methods : 1. It was made by cutting a TLM2 alloy plate and drilling tiny holes with laser on it. Rat NIH-3T3 cells were plated at a density of 1×105cells/ml on porous TLM samples placed in 6-well plastic plates after high pressure sterilization. Then they were cultured for 4 hours .The material was scaned by an electron microscope. The microscopic photos were analyzed by image pattern analysis to measure the porous size and porous rate.The cells on the samples surface were observed to assess its activit and shape. 2. Porous TLM alloy was used as a tissue scaffold for airway regeneration in five dogs. the larynx and the trachea were exposed. The anterior 1/3 part of the cricoid cartilage (cover the cricothyroid membrane) and 1 cervical trachea were simultaneously resected by cold instruments form an artificial anterior larynx open. The“◇”scaffold material was implanted into the resultant defect. Endoscopic examination was undertaken periodically for a postoperative period of 3 to 6 months in all dogs.3. A porous type of cricotracheal prosthesis made of porous TLM alloy is designed. This prosthesis (20 mm in length) is used to reconstruct the cricoid cartilage and the first tracheal ring in 10 mongrel dogs and its efficacy is evaluated. 4. Twenty adult mongrel dogs were chosen , divided randomly two groups. The TLM prosthesis were implanted into one group of dogs(n=10),and the pure titanium(Ti-2) prosthesis were implanted into another group of dogs(n=10). Cricoid cartilage and the first 1,2 tracheal ring reconstruction was performed with the biomedical materials both the TLM and the Ti-2 prosthesis (25 mm in length) in dogs. All dogs were euthanized in 1, 4 and 12 weeks and tracheal CT scan, tracheal endoscopy,histopathological examination and scanning electron microscopic examinations were performed. 5. 5 cm long segment of the cervical trachea was resected in 5 dogs. Porous TLM is fabricated into the shape of a human trachea to repair circumferential tracheal defects. The planned period for the implantation was from3 to 8 months before the sacrifice of the animals for endoscopic examination, CT scan and histological analysis.
Results: 1. Scanning electron microscopic photos showed that the surface structure of the samples was porous and the diameters varied from 70 to 90μm.The image analysis demonstrated that the porous rate was 32%. Cells appeared to have a normal cellular shape with lamellipodia and pseudopodia. 2. Endoscopic examination showed the implant located perfectly and the airway is wide enough for breath. There was no airway stenosis in any of the dogs. Granulation was observed in no cases, and anastomotic dehiscence was not observed. 3. One dog died due to an accident with anesthesia at 1 week, one dog died of pneumonia about 1 month after operation,and six dogs were killed between 3 and 8 months; At sacrifice, all the prostheses had become completely incorporated into the host tissue. Endoscopic examination showed no airway obstruction for a postoperative period of 3 to 8 months in 8 dogs. Granulation was observed in 4 cases, and the porous TLM alloy plate was exposed in 1 case, but these dogs were asymptomatic. Light microscopy and electron microscopy showed a normal mucous membrane growth on the prosthesis surface and no visible granulation tissue in the reconstructed cricoid and tracheal cavity. 4. Two type of the biomedical materials showed excellent mechanical strength after implantation. the Porous TLM alloy prothesis was present air-tightness after implantation at early period. The CT scan,endoscopic examination showed no apparent airway obstruction for a postoperative period of 1 to 12 weeks in TLM group,only 3 cases was free of airway obstruction in Ti-2 group. Histologic result showed a normal mucous membrane with normal cilial growth on the TLM implant.5. The mechanical performance of the prostheses was good and the protheses were integrated by the surrounding tissue.Results showed an adequate consistency of prosthesis, adequate tolerance without producing tracheal stenoses, and impermeability to air, allowing a correct invasion by granulation tissue.
Conclusion : 1. For the first time, we conclude that this TLM alloy is a kind of excellent biomedical material in replacement of larynx and tracheal defect due to its traits as having moderate strength,toughness matching,low elastic modulus(30～50Gpa),corrosion resistance,which provide a good stress distribution for reconstruction of laryngotracheal defect. 2. For the first time, It was made by cutting a TLM alloy plate and drilling tiny holes with laser on it. The diameter of the porosities was 70~90μm,giving a porosity of 30~35%, thickness was 0.5mm.We found that good incorporation of the prosthesis pore into the host tissue to maintain the air-tightness of the prosthesis after implantation in the early period,and confluent regeneration of the epithelium over the TLM alloy to overcome stenosis of prosthesis lumen. This process was faster than any found in any other porous tracheal prosthesis so far tested. We further proved that epithelialization results from capillary invasion through the prosthetic pores and growth from both tracheal ends.3. The TLM alloy is a potential new biomaterial for laryngotracheal defect reconstruction,which has highly research value for clinical application in the future.
引文
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