Masquelet技术联合人工真皮对家兔骨与软组织缺损的治疗研究
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摘要
目的探讨应用Masquelet技术联合人工真皮对家兔骨与软组织缺损的修复效果,观察诱导膜的微观结构及血管化情况,以期指导临床中对Gustilo-Anderson Ⅲ型开放性骨折伴大段骨缺损及软组织缺损的治疗。方法雄性家兔80只,体质量2.03～2.27 kg,平均2.11 kg。取64只兔双侧大腿随机分为实验组及对照组,余16只兔双侧为假手术组。所有兔均制备股骨骨质缺损及软组织缺损模型。实验组以Masquelet技术第一阶段方法[将聚甲基丙烯酸甲酯骨水泥填充于骨缺损区]联合人工真皮治疗;对照组单纯以Masquelet技术第一阶段方法治疗;假手术组不作处理,直接缝合伤口。术后2、4、6、8周各处死假手术组4只家兔,以及实验组/对照组16只家兔,于双侧股骨原手术部位取材,大体观察诱导膜及结合膜情况;对膜结构取材行常规HE染色观察其微观结构;行CD34免疫组织化学染色观察,并计数微血管密度(microvessel density,MVD)。结果大体观察示:术后4周,实验组人工真皮胶原蛋白海绵层完全消失,实验组及对照组均产生完整诱导膜结构;对照组膜结构呈半透明状,实验组膜结构较厚,呈淡红色不透明,伴小血管增生。术后6～8周,实验组和对照组膜结构均逐渐增厚。假手术组随时间变化仅观察到瘢痕组织增生。HE染色示:术后2周,实验组及对照组均可见大量肌纤维,少量胶原纤维增生伴炎性细胞浸润;假手术组大多为肌纤维,伴少量肌纤维间血管。术后4周,实验组较对照组胶原纤维增多,部分血管形成,对照组胶原纤维细胞核呈类圆形,实验组呈扁圆形。术后6、8周实验组和对照组胶原纤维均较前增多,对照组胶原纤维细胞核仍呈类圆形,实验组呈梭形且细胞核较同期对照组深染;两组均可观察到增生血管,实验组增生血管数目较对照组增多。假手术组仍可见大量成纤维细胞出现,未表现随时间变化的血管显著增生。CD34免疫组织化学染色示,术后随时间延长,各组MVD均呈逐渐增加趋势。术后2周假手术组MVD显著大于实验组和对照组,4、6、8周显著小于实验组和对照组,差异均有统计学意义(P<0.05)。实验组术后4、6周MVD显著大于对照组(P<0.05),2、8周两组MVD比较差异无统计学意义(P>0.05)。结论 Masquelet技术结合人工真皮治疗兔股骨骨质缺损及软组织缺损,在术后4～6周明显促进了膜结构的血管化过程。这两种方法结合对临床Gustilo-AndersonⅢ型开放性骨折伴骨及软组织缺损的治疗有指导意义。
Objective To investigate the effect of Masquelet technique combined with artificial dermis on repairing bone and soft tissue defects in rabbits, and to observe the microstructure and vascularization of induced membrane, so as to guide the clinical treatment of Gustilo-Anderson type Ⅲ open fracture with large bone defect and soft tissue defect. Methods Eighty male rabbits, weighing 2.03-2.27 kg(mean, 2.11 kg), were selected. The bilateral thighs of64 rabbits were randomly divided into experimental group and control group, the remaining 16 rabbits were sham operation group. Bone and soft tissue defect models of femur were made in all rabbits. In the experimental group, the first stage of Masquelet technique was used [polymethyl methacrylate bone cement was filled in bone defect area] combined with artificial dermis treatment; in the control group, the first stage of Masquelet technique was used only; in the sham operation group, the wound was sutured directly without any treatment. Four rabbits in sham operation group and 16 rabbits in the experimental group and control group were sacrificed at 2, 4, 6, and 8 weeks after operation, respectively.The induced membranes and conjunctive membranes were observed on both sides of the femur. The membrane structure was observed by HE staining, and the microvessel density(MVD) was counted by CD34 immunohistochemical staining.Results Gross observation showed that the spongy layer of collagen in the artificial dermis of the experimental group disappeared completely at 4 weeks after operation, and the induced membrane structure of the experimental group and the control group was complete; the membrane structure of the control group was translucent, and the membrane structure of the experimental group was thicker, light red opaque, accompanied by small vessel proliferation. The membrane structure of the experimental group and the control group increased gradually from 6 to 8 weeks after operation. In the sham operation group, only scar tissue proliferation was observed over time. HE staining showed that a large number of muscle fibers and a small amount of collagen fibers proliferation with inflammatory cell infiltration could be seen in the experimental group and the control group at 2 weeks after operation; most of the sham operation group were muscle fibers with a small amount of interfibrous vessels. At 4 weeks after operation, collagen fibers increased and some blood vessels formed in the experimental group. The nuclei of collagen fibers in the control group were round-like,while those in the experimental group were flat-round. At 6 and 8 weeks after operation, the collagen fibers in the experimental group and the control group increased. The nuclei of the collagen fibers in the control group were still round-like. The nuclei of the collagen fibers in the experimental group were fusiformis and deeply stained compared with those in the control group. The proliferation of blood vessels was observed in both groups, and the number of proliferation vessels in the experimental group was increased compared with that in the control group. In the sham operation group, a large number of fibroblasts still appeared, but no significant proliferation of blood vessels with time was observed. CD34 immunohistochemical staining showed that MVD in each group increased gradually with the prolongation of time after operation. MVD in the sham operation group was significantly higher than that in the experimental group and the control group at 2 weeks after operation, and significantly smaller than that in the experimental group and the control group at 4, 6, and 8 weeks after operation(P<0.05). MVD in the experimental group was significantly higher than that in the control group at 4 and 6 weeks after operation(P<0.05), but there was no significant difference in MVD between the two groups at 2 and 8 weeks(P>0.05). Conclusion Masquelet technique combined with artificial dermis in the treatment of femoral bone defect and soft tissue defect in rabbits can significantly promote the vascularization of membrane structure at 4-6 weeks after operation. The combination of these two methods has guiding significance for the treatment of Gustilo-Anderson type Ⅲ open fracture with bone and soft tissue defects.
Objective To investigate the effect of Masquelet technique combined with artificial dermis on repairing bone and soft tissue defects in rabbits, and to observe the microstructure and vascularization of induced membrane, so as to guide the clinical treatment of Gustilo-Anderson type Ⅲ open fracture with large bone defect and soft tissue defect. Methods Eighty male rabbits, weighing 2.03-2.27 kg(mean, 2.11 kg), were selected. The bilateral thighs of64 rabbits were randomly divided into experimental group and control group, the remaining 16 rabbits were sham operation group. Bone and soft tissue defect models of femur were made in all rabbits. In the experimental group, the first stage of Masquelet technique was used [polymethyl methacrylate bone cement was filled in bone defect area] combined with artificial dermis treatment; in the control group, the first stage of Masquelet technique was used only; in the sham operation group, the wound was sutured directly without any treatment. Four rabbits in sham operation group and 16 rabbits in the experimental group and control group were sacrificed at 2, 4, 6, and 8 weeks after operation, respectively.The induced membranes and conjunctive membranes were observed on both sides of the femur. The membrane structure was observed by HE staining, and the microvessel density(MVD) was counted by CD34 immunohistochemical staining.Results Gross observation showed that the spongy layer of collagen in the artificial dermis of the experimental group disappeared completely at 4 weeks after operation, and the induced membrane structure of the experimental group and the control group was complete; the membrane structure of the control group was translucent, and the membrane structure of the experimental group was thicker, light red opaque, accompanied by small vessel proliferation. The membrane structure of the experimental group and the control group increased gradually from 6 to 8 weeks after operation. In the sham operation group, only scar tissue proliferation was observed over time. HE staining showed that a large number of muscle fibers and a small amount of collagen fibers proliferation with inflammatory cell infiltration could be seen in the experimental group and the control group at 2 weeks after operation; most of the sham operation group were muscle fibers with a small amount of interfibrous vessels. At 4 weeks after operation, collagen fibers increased and some blood vessels formed in the experimental group. The nuclei of collagen fibers in the control group were round-like,while those in the experimental group were flat-round. At 6 and 8 weeks after operation, the collagen fibers in the experimental group and the control group increased. The nuclei of the collagen fibers in the control group were still round-like. The nuclei of the collagen fibers in the experimental group were fusiformis and deeply stained compared with those in the control group. The proliferation of blood vessels was observed in both groups, and the number of proliferation vessels in the experimental group was increased compared with that in the control group. In the sham operation group, a large number of fibroblasts still appeared, but no significant proliferation of blood vessels with time was observed. CD34 immunohistochemical staining showed that MVD in each group increased gradually with the prolongation of time after operation. MVD in the sham operation group was significantly higher than that in the experimental group and the control group at 2 weeks after operation, and significantly smaller than that in the experimental group and the control group at 4, 6, and 8 weeks after operation(P<0.05). MVD in the experimental group was significantly higher than that in the control group at 4 and 6 weeks after operation(P<0.05), but there was no significant difference in MVD between the two groups at 2 and 8 weeks(P>0.05). Conclusion Masquelet technique combined with artificial dermis in the treatment of femoral bone defect and soft tissue defect in rabbits can significantly promote the vascularization of membrane structure at 4-6 weeks after operation. The combination of these two methods has guiding significance for the treatment of Gustilo-Anderson type Ⅲ open fracture with bone and soft tissue defects.
引文
1 Masquelet AC, Fitoussi F, Begue T, et al. Reconstruction of thelong bones by the induced membrane and spongy autograft. Ann Chir Plast Esthet, 2000, 45(3):346-353.
2 Masquelet AC, Begue T. The concept of induced membrane for reconstruction of long bone defects. Orthop Clin North Am, 2010,41(1):27-37.
3 Chen X, Chen H, Zhang G. Management of wounds with exposed bone structures using an artificial dermis and skin grafting technique. Clin Plast Surg, 2012, 39(1):69-75.
4 Molnar JA, DeFranzo AJ, Hadaegh A, et al. Acceleration of Integra incorporation in complex tissue defects with subatmospheric pressure. Plast Reconstr Surg, 2004, 113(5):1339-1346.
5 Lee LF, Porch JV, Spenler W, et al. Integra in lower extremity reconstruction after burn injury. Plast Reconstr Surg, 2008, 121(4):1256-1262.
6 Clerici G, Caminiti M, Curci V, et al. The use of a dermal substitute to preserve maximal foot length in diabetic foot wounds with tendon and bone exposure following urgent surgical debridement for acute infection. Int Wound J, 2010, 7(3):176-183.
7 Yeong EK, Yu YC, Chan ZH, et al. Is artificial dermis an effective tool in the treatment of tendon-exposed wounds. J Burn Care Res,2013,34(1):161-167.
8 Murray RC, Gordin EA, Saigal K, et al. Reconstruction radial forearm free flap donor site using integra artificial dermis.Microsurgery, 2011, 31(2):104-108.
9 Yeong EK, Chen SH, Tang YB. The treatment of bone exposure in burns by using artificial dermis. Ann Plast Surg, 2012, 69(6):607-610.
10 Tarchala M, Harvey EJ, Barralet J. Biomaterial-stabilized soft tissue healing for healing of critical-sized bone defects:the Masquelet technique. Adv Healthc Mater, 2016, 5(6):630-640.
11 Wiese A, Pape HC. Bone defects caused by high-energy injuries,bone loss, infected nonunions, and nonunions. Orthop Clin North Am, 2010,41(1):1-4.
12 Viateau V, Guillemin G, Calando Y, et al. Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects:an ovine model. Vet Surg, 2006, 35(5):445-452.
13 Precheur HV. Bone graft materials. Dent Clin North Am, 2007,51(3):729-746.
14 Wang F, Wang M, She Z, et al. Collagen/chitosan based two compartment and bi-functional dermal scaffolds for skin regeneration. Mater Sci Eng C Mater Biol Appl, 2015, 52:155-162.
15 Giannoudis PV, Faour O, Goff T, et al. Masquelet technique for the treatment of bone defects:tips-tricks and future directions. Injury,2011,42(6):591-598.
16 Mekhail AO, Abraham E, Gruber B, et al. Bone transport in the management of posttraumatic bone defects in the lower extremity.J Trauma, 2004, 56(2):368-378.
17 Pelissier P, Martin D, Baudet J, et al. Behaviour of cancellous bone graft placed in induced membranes. Br J Plast Surg, 2002, 55(7):596-598.
18 Myeroff C, Archdeacon M. Autogenous bone graft:donor sites and techniques. J Bone Joint Surg(Am), 2011, 93(23):2227-2236.
19张磊,王新卫,陈雨声,等.应用Ilizarov技术治疗骨髓炎出现复杂软组织问题的处理技巧探讨.中国修复重建外科杂志,2018,32(10):1286-1291.
20 Motsitsi NS. Masquelet's technique for management of long bone defects:from experiment to clinical application. East Cent Afr JSurg, 2012, 17(2):43-47.
21 Henrich D, Seebach C, Nau C, et al. Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical-sized defect model. J Tissue Eng Regen Med,2016,10(10):E382-E396.
22 Koga Y, Komuro Y, Yamato M, et al. Recovery course of fullthickness skin defects with exposed bone:an evaluation by a quantitative examination of new blood vessels. J Surg Res, 2007,137(1):30-37.
23沈余明,陈辉,胡骁骅,等.组织瓣移植联合骨延长技术分期修复烧创伤后下肢严重软组织与骨缺损.中国修复重建外科杂志,2017,31(2):160-164.
24 Dantzer E, Braye FM. Reconstructive surgery using an artificial dermis(Integra):results with 39 grafts. Br J Plast Surg, 2001, 54(8):659-664.
25 Burke JF, Yannas IV, Quinby WC Jr, et al. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg, 1981, 194(4):413-428.
26 Suzuki S, Matsuda K, Maruguchi T, et al. Further applications of"bilayer artificial skin". Br J Plast Surg, 1995, 48(4):222-229.
27 Wosgrau AC, Jeremias Tda S, Leonardi DF, et al. Comparative experimental study of wound healing in mice:pelnac versus integra. PLoS One, 2015, 10(3):e0120322.
28 Jeremias Tad S, Machado RG, Visoni SB, et al. Dermal substitutes support the growth of human skin-derived mesenchymal stromal cells:potential tool for skin regeneration. PLoS One, 2014, 9(2):e89542.
29 Wong TM, Lau TW, Li X, et al. Masquelet technique for treatment of posttraumatic bone defects. ScientificWorldJournal, 2014, 2014:710302.
30 Gouron R, Petit L, Boudot C, et al. Osteoclasts and their precursors are present in the induced-membrane during bone reconstruction using the Masquelet technique. J Tissue Eng Regen Med, 2017,11(2):382-389.
31 Aho OM, Lehenkari P, Ristiniemi J, et al. The mechanism of action of induced membranes in bone repair. J Bone Joint Surg(Am),2013, 95(7):597-604.
32 Woon CY, Chong KW, Wong MK. Induced membranes-a staged technique of bone-grafting for segmental bone loss:a report of two cases and a literature review. J Bone Joint Surg(Am), 2010, 92(1):196-201.
33 Pelissier P, Masquelet AC, Bareille R, et al. Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration. J Orthop Res, 2004,22(1):73-79.
34 Masquelet AC. Muscle reconstruction in reconstructive surgery:soft tissue repair and long bone reconstruction. Langenbecks Arch Surg, 2003, 388(5):344-346.
35 Christou C, Oliver RA, Yu Y, et al. The Masquelet technique for membrane induction and the healing of ovine critical sized segmental defects. PLoS One, 2014, 9(12):e114122.
36 Kenneth Ward W. A review of the foreign-body response to subcutaneously-implanted devices:the role of macrophages and cytokines in biofouling and fibrosis. J Diabetes Sci Technol, 2008,2(5):768-777.
2 Masquelet AC, Begue T. The concept of induced membrane for reconstruction of long bone defects. Orthop Clin North Am, 2010,41(1):27-37.
3 Chen X, Chen H, Zhang G. Management of wounds with exposed bone structures using an artificial dermis and skin grafting technique. Clin Plast Surg, 2012, 39(1):69-75.
4 Molnar JA, DeFranzo AJ, Hadaegh A, et al. Acceleration of Integra incorporation in complex tissue defects with subatmospheric pressure. Plast Reconstr Surg, 2004, 113(5):1339-1346.
5 Lee LF, Porch JV, Spenler W, et al. Integra in lower extremity reconstruction after burn injury. Plast Reconstr Surg, 2008, 121(4):1256-1262.
6 Clerici G, Caminiti M, Curci V, et al. The use of a dermal substitute to preserve maximal foot length in diabetic foot wounds with tendon and bone exposure following urgent surgical debridement for acute infection. Int Wound J, 2010, 7(3):176-183.
7 Yeong EK, Yu YC, Chan ZH, et al. Is artificial dermis an effective tool in the treatment of tendon-exposed wounds. J Burn Care Res,2013,34(1):161-167.
8 Murray RC, Gordin EA, Saigal K, et al. Reconstruction radial forearm free flap donor site using integra artificial dermis.Microsurgery, 2011, 31(2):104-108.
9 Yeong EK, Chen SH, Tang YB. The treatment of bone exposure in burns by using artificial dermis. Ann Plast Surg, 2012, 69(6):607-610.
10 Tarchala M, Harvey EJ, Barralet J. Biomaterial-stabilized soft tissue healing for healing of critical-sized bone defects:the Masquelet technique. Adv Healthc Mater, 2016, 5(6):630-640.
11 Wiese A, Pape HC. Bone defects caused by high-energy injuries,bone loss, infected nonunions, and nonunions. Orthop Clin North Am, 2010,41(1):1-4.
12 Viateau V, Guillemin G, Calando Y, et al. Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects:an ovine model. Vet Surg, 2006, 35(5):445-452.
13 Precheur HV. Bone graft materials. Dent Clin North Am, 2007,51(3):729-746.
14 Wang F, Wang M, She Z, et al. Collagen/chitosan based two compartment and bi-functional dermal scaffolds for skin regeneration. Mater Sci Eng C Mater Biol Appl, 2015, 52:155-162.
15 Giannoudis PV, Faour O, Goff T, et al. Masquelet technique for the treatment of bone defects:tips-tricks and future directions. Injury,2011,42(6):591-598.
16 Mekhail AO, Abraham E, Gruber B, et al. Bone transport in the management of posttraumatic bone defects in the lower extremity.J Trauma, 2004, 56(2):368-378.
17 Pelissier P, Martin D, Baudet J, et al. Behaviour of cancellous bone graft placed in induced membranes. Br J Plast Surg, 2002, 55(7):596-598.
18 Myeroff C, Archdeacon M. Autogenous bone graft:donor sites and techniques. J Bone Joint Surg(Am), 2011, 93(23):2227-2236.
19张磊,王新卫,陈雨声,等.应用Ilizarov技术治疗骨髓炎出现复杂软组织问题的处理技巧探讨.中国修复重建外科杂志,2018,32(10):1286-1291.
20 Motsitsi NS. Masquelet's technique for management of long bone defects:from experiment to clinical application. East Cent Afr JSurg, 2012, 17(2):43-47.
21 Henrich D, Seebach C, Nau C, et al. Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical-sized defect model. J Tissue Eng Regen Med,2016,10(10):E382-E396.
22 Koga Y, Komuro Y, Yamato M, et al. Recovery course of fullthickness skin defects with exposed bone:an evaluation by a quantitative examination of new blood vessels. J Surg Res, 2007,137(1):30-37.
23沈余明,陈辉,胡骁骅,等.组织瓣移植联合骨延长技术分期修复烧创伤后下肢严重软组织与骨缺损.中国修复重建外科杂志,2017,31(2):160-164.
24 Dantzer E, Braye FM. Reconstructive surgery using an artificial dermis(Integra):results with 39 grafts. Br J Plast Surg, 2001, 54(8):659-664.
25 Burke JF, Yannas IV, Quinby WC Jr, et al. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg, 1981, 194(4):413-428.
26 Suzuki S, Matsuda K, Maruguchi T, et al. Further applications of"bilayer artificial skin". Br J Plast Surg, 1995, 48(4):222-229.
27 Wosgrau AC, Jeremias Tda S, Leonardi DF, et al. Comparative experimental study of wound healing in mice:pelnac versus integra. PLoS One, 2015, 10(3):e0120322.
28 Jeremias Tad S, Machado RG, Visoni SB, et al. Dermal substitutes support the growth of human skin-derived mesenchymal stromal cells:potential tool for skin regeneration. PLoS One, 2014, 9(2):e89542.
29 Wong TM, Lau TW, Li X, et al. Masquelet technique for treatment of posttraumatic bone defects. ScientificWorldJournal, 2014, 2014:710302.
30 Gouron R, Petit L, Boudot C, et al. Osteoclasts and their precursors are present in the induced-membrane during bone reconstruction using the Masquelet technique. J Tissue Eng Regen Med, 2017,11(2):382-389.
31 Aho OM, Lehenkari P, Ristiniemi J, et al. The mechanism of action of induced membranes in bone repair. J Bone Joint Surg(Am),2013, 95(7):597-604.
32 Woon CY, Chong KW, Wong MK. Induced membranes-a staged technique of bone-grafting for segmental bone loss:a report of two cases and a literature review. J Bone Joint Surg(Am), 2010, 92(1):196-201.
33 Pelissier P, Masquelet AC, Bareille R, et al. Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration. J Orthop Res, 2004,22(1):73-79.
34 Masquelet AC. Muscle reconstruction in reconstructive surgery:soft tissue repair and long bone reconstruction. Langenbecks Arch Surg, 2003, 388(5):344-346.
35 Christou C, Oliver RA, Yu Y, et al. The Masquelet technique for membrane induction and the healing of ovine critical sized segmental defects. PLoS One, 2014, 9(12):e114122.
36 Kenneth Ward W. A review of the foreign-body response to subcutaneously-implanted devices:the role of macrophages and cytokines in biofouling and fibrosis. J Diabetes Sci Technol, 2008,2(5):768-777.