Transplantation of Autologous Tissue Fragments into an e-PTFE Graft with Long Fibrils

Abstract: An expanded polytetrafluoroethylene (e-PTFE) graft with long fibrils transplanted with bone marrow showed rapid and uniform neointima formation in a dog study. The e-PTFE grafts (fibril length, 90 μm; 6 mm internal diameter; length, 6–8 cm) transplanted with autologous bone marrow were implanted in the abdominal aortae of 10 dogs and retrieved at 3 weeks and 3 months after implantation. Control e-PTFE grafts without bone marrow treatment were also implanted in the same manner in 8 dogs. Macroscopically the treated graft wall appeared red in color; however, there was no thrombus deposition on the surface. Light microscopic observation revealed that the treated grafts were completely lined with endothelial cells at 3 weeks. The neointima was uniform without intimai hyperplasia at the anastomotic sites.
Inside the graft wall many capillary blood vessels were observed. At 3 months moderate intimai hyperplasia throughout the graft with complete endothelialization was observed. In the control grafts, endothelialization was observed at the anastomotic sites; however, half of the other areas were covered with a fibrin layer devoid of endothelial cells even in the 3-month grafts. These results indicated that neointima formation was effectively accelerated with the autologous bone marrow transplantation, but moderate intimai hyperplasia throughout the graft was inevitable in e-PTFE grafts even after complete endothelialization.     

ePTFE人工血管材料内衬自体骨髓内皮细胞的实验研究

目的:探讨自体骨髓内皮细胞衬里对ePTFE人工血管材料通畅性的影响。方法:24只犬随机分入实验组(12只)和对照组(12只)。实验组取犬自体骨髓,提取单核细胞种植ePTFE人工血管,行左髂动脉人工血管置换术;对照组采用普通ePTFE人工血管。分别于术后1月、3月行彩色多普勒超声观察其通畅率,并行组织染色及扫描电镜观察,比较人工血管表面内皮化情况。结果:术后1月时实验组人工血管通畅率及内膜厚度同对照组均无明显差别;但术后3月时实验组通畅率高于对照组,内膜厚度明显小于对照组(P0.05)。结论:自体骨髓内皮细胞衬里技术可明显提高ePTFE人工血管的短期通畅率,但其对远期通畅率的影响尚有待进一步观察。     

Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow.

Hydroxyapatite is a synthetic bone graft, which is used for the treatment of bone defects and nonunions. However, it is a rather inert material with no or little intrinsic osteoinductive activity. Recombinant human osteogenic protein-1 (rhOP-1) is a very potent biological agent, that enhances osteogenesis during bone repair. Bone marrow contains mesenchymal stem cells, which are capable of new bone formation. Biosynthetic bone grafts were created by the addition of rhOP-1 or bone marrow to granular porous hydroxyapatite. The performance of these grafts was tested in a sheep model and compared to the results of autograft, which is clinically the standard treatment of bone defects and nonunions. A 3 cm segmental bone defect was made in the tibia and fixed with an interlocking intramedullary nail. There were five treatment groups: no implant (n=6), autograft (n=8), hydroxyapatite alone (n=8), hydroxyapatite loaded with rhOP-1 (n=8), and hydroxyapatite loaded with autologous bone marrow (n=8). At 12 weeks, healing of the defect was evaluated with radiographs, a torsional test to failure, and histological examination of longitudinal sections through the defect. Torsional strength and stiffness of the healing tibiae were about two to three times higher for autograft and hydroxyapatite plus rhOP-1 or bone marrow compared to hydroxyapatite alone and empty defects. The mean values of both combination groups were comparable to those of autograft. There were more unions in defects with hydroxyapatite plus rhOP-1 than in defects with hydroxyapatite alone. Although the differences were not significant, histological examination revealed that there was more often bony bridging of the defect in both combination groups and the autograft group than in the group with hydroxyapatite alone. Healing of bone defects, treated with porous hydroxyapatite, can be enhanced by the addition of rhOP-1 or autologous bone marrow. The results of these composite biosynthetic grafts are equivalent to those of autograft.     

Comparison of prosthetic graft materials as intracardiac right atrial patches

An experimental animal model was developed to study the fate of prosthetic graft materials within the heart. Autologous pericardium, bovine pericardium, polytetrafluoroethylene, woven Dacron, and autologous right atrium (control) patches were implanted into the wall at three sites on the right atrium in each of 10 dogs (six patches for each graft material). The atria were harvested 90-100 days later and histologic examination and quantitation of calcium were performed. Chronic inflammation and fibrosis were found in 29 of 30 grafts. Cartilage formed in 26 of 30 sites and was found both in the center and around the edges of the grafts. In addition to cartilage, bone including marrow elements formed in the two autologous materials at 4 of the 12 sites and in 1 of the Dacron graft sites. Calcium content was greater in the control and the bovine pericardial grafts than in the other graft materials (P = NS). The incidence and degree of inflammation, fibrosis, calcification and cartilage, and bone formation were similar in all materials. We conclude that the healing process of these intracardiac graft materials is a generalized phenomenon independent of the inherent properties of a specific graft material.     

Combined use of platelet-rich plasma and autologous bone grafts in the treatment of long bone defects in mini-pigs

The use of platelet-rich plasma (PRP) for improving of bone defect healing is discussed controversially. The aim of this study was to assess the effect of PRP in combination with autologous cancellous graft on bone defect healing in a critical metaphyseal long bone defect. A critical size defect in the tibial metaphysis of 16 mini-pigs was filled either with autologous cancellous graft as control group or with autologous cancellous graft combined with autologous PRP. Compared to native blood platelets were enriched about 4.9-fold in the PRP. After 6 weeks, the specimens were assessed by X-ray and histological evaluation. Histomorphometrical analysis revealed that the area of new bone was significantly higher in the PRP group concerning the central area of the defect zone (p < 0.02) as well as the cortical defect zone (p < 0.01). All defects showed substantial new bone formation, but only defects of the PRP group regenerated entirely. The PRP group was superior to the control group even in the semi-quantitative assessment of the osseous bridging in both observed areas of the defect. Within the limits of the present study it could be demonstrated that PRP combined with autologous cancellous graft leads to a significantly better bone regeneration compared to isolated application of autologous cancellous graft in an in vivo critical size defect on load-bearing long bones of mini-pigs.     

Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein-7

Growth factors contained in platelet-rich plasma (PRP) have recently been proposed to enhance maturation of bone grafts and, in combination with anorganic bovine bone, to support repair in the treatment of small bone defects in maxillofacial surgery. Bone morphogenetic proteins (BMP) carried in a matrix may be able to replace the autologous bone graft in the treatment of critical size defects. However, no studies have compared the bone stimulating capacity of PRP and BMP. Likewise there is no data comparing the effects of PRP in either an autologous bone graft or in anorganic bovine bone. We augmented the mandible of Wistar rats (n = 28) on both sides with either anorganic bovine bone (Bio-Oss) or autologous rib bone. On the test side we applied either 20 microl of autologous PRP or 10 microl of rhBMP-7 (4 groups, n = 7). In addition, bone induction was evaluated in an extraskeletal site (n = 14). A polychrome sequential labeling was performed. The animals were sacrificed by intra-vital perfusion on day 50. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. The qualitative analysis of fluorochrome labels suggested that PRP and rhBMP-7 accelerated bone growth. However, histomorphometric analysis revealed no significant differences in the area of newly mineralized bone under either the influence of PRP or rhBMP-7 on autologous bone graft. Likewise, the addition of PRP to anorganic bovine bone showed no statistical difference to the control group. The strongest bone stimulating effect was seen for the combination of rhBMP-7 with anorganic bovine bone (p = 0.028). In the extraskeletal model, newly formed bone was evident in the presence of rhBMP-7, but not of PRP. In conclusion, according to the histomorphometry, the addition of platelet-rich plasma failed to enhance bone formation on anorganic bovine bone and on autologous bone grafts.     

Promising cell-based therapy for bone regeneration using stem cells from deciduous teeth, dental pulp, and bone marrow

We attempted to regenerate bone in a significant osseous defect with various stem cells from deciduous teeth, extracted from puppies, and grafted them into a parent canine mandible as an allograft, parent dental pulp, and bone marrow by tissue engineering and regenerative medicine technology using platelet-rich plasma as an autologous scaffold and signal molecules. Initially, teeth were extracted from a child and parent hybrid canine mandible region and bone marrow (canine mesenchymal stem cells; cMSCs), and parent teeth (canine dental pulp stem cells; cDPSCs), and stem cells were extracted from deciduous teeth (puppy deciduous teeth stem cells; pDTSCs). After 4 weeks, bone defects were prepared on both sides of the mandible with a trephine bar. Graft materials were implanted into these defects: 1) control (defect only), 2) platelet-rich plasma (PRP), 3) cMSCs/PRP, 4) cDPSCs/PRP, and 5) pDTSCs/PRP to investigate the effect of stem cells. The newly formed bones were evaluated by histology and histomorphometric analysis in the defects at 2, 4, and 8 weeks. According to histological observations, the cMSCs/PRP, cDPSCs/PRP, and pDTSCs/PRP groups had well-formed mature bone and neovascularization compared with the control (defect only) and PRP groups at 4 and 8 weeks, respectively, and the mineralized tissues in cMSCs/PRP, cDPSCs/PRP, and pDTSCs/PRP specimens were positive for osteocalcin at 8 weeks. Histometrically, newly formed bone areas were 19.0 ± 2.9% (control), 19.7 ± 6.0% (PRP), 52.8 ± 3.5% (cMSCs/PRP), 61.6 ± 1.3% (cDPSCs/PRP), and 54.7 ± 2.2% (pDTSCs/PRP) at 8 weeks. There were significant differences between cMSCs, cDPSCs, pDTSCs/PRP, and control and PRP groups. These results demonstrate that stem cells from deciduous teeth, dental pulp, and bone marrow with PRP have the ability to form bone, and bone formation with DTSCs might have the potential to generate a graft between a child and parent. This preclinical study could pave the way for stem cell therapy in orthopedics and oral maxillofacial reconstruction for clinical application.     

骨髓干细胞复合纳米骨用于脊柱融合的研究

目的 探讨骨髓间充质干细胞(BMSCs)复合纳米羟基磷灰石/胶原(nHAC)用于脊柱融合的可行性.方法 将20只新西兰大白兔根据双侧L5～L6横突间植入物不同分为复合材料组和自体髂骨组,每组10只.术后4周每组取材2只,术后8周取材剩余兔,行X线、大体观察、手触检测和组织学观察,评估腰椎融合情况.结果术后8周复合材料组和自体髂骨组融合率分别为75.0%(6/8)和87.5%(7/8),两组融合率差异无统计学意义(P>0.05).结论 BMSCs复合nHAC是一种较好的植骨替代材料,用于脊柱融合可获得与自体骨移植近似地融合效果.     

构建猕猴组织工程化周围神经的实验研究

目的评价组织工程化周围神经修复猕猴4cm尺神经缺损的实验效果,为临床研究提供资料。方法分别用6种移植物桥接4cm尺神经缺损。A组:自体BMSCs 去细胞同种异体神经支架;B组:自体SCs 去细胞同种异体神经支架;C组:自体BMSCs PLGA支架导管;D组:去细胞同种异体神经支架;E组:PLGA支架导管;F组:自体神经。通过功能学、神经电生理学及组织学研究评价各自的实验效果。结果A、B、C三种组织工程化神经实验组,术后6个月神经电生理和组织学检查,能引起小鱼际肌群产生复合动作电位的潜伏期、复合动作电位的最大振幅、神经传导速度和再生的神经纤维数目与自体神经移植组(F组)相比差异无显著性意义(P>0.05),但分别大于未加细胞的支架组(D、E组),差异有显著意义(P<0.05)。结论用自体源SCs或BMSCs作种子细胞与去细胞同种异体神经支架,或自体源BMSCs与PLGA支架导管构建不同的组织工程化周围神经,修复猕猴4cm尺神经缺损均取得较好的效果。     

Long-bone critical-size defects treated with tissue-engineered grafts: a study on sheep.

Standardized particulate bone constructs, obtained by expanding autologous mesenchymal stem cells (MSCs) onto coral granules in vitro, were transplanted into long-bone, critical-size defects in sheep. Control experiments were also performed in which autologous bone grafts were implanted. Defect cavities were lined with a preformed vascularized membrane (induced by temporarily inserting a cement spacer for 6 weeks prior to bone construct implantation), which served as a mold keeping the engineered bone granules in place. Radiographic, histological, and computed tomographic tests performed 6 months later showed that the osteogenic abilities of the engineered construct and autograft were significantly greater than those of coral scaffold alone. No significant differences were found between the amount of newly formed bone in defects filled with coral/MSCs and those filled with autograft, yet radiological scores differed significantly between the two groups (21% and 100% healed cortices, respectively). The present study on a clinically relevant animal model provides the first evidence that standardized particulate bone constructs can be used to repair large bone defects and that their osteogenic ability approaches that of bone autograft, the bone repair benchmark. By proving feasibility, the present study makes possible the treatment of segmental bone losses with bone constructs engineered from granules, a process which is much simpler than preparing customized massive constructs using computer-assisted techniques. Important parameters, such as the rate of scaffold resorption and the number of MSCs to be seeded on the scaffolds, need to be optimized before reaching pertinent definitive conclusions.     

Translational research for injectable tissue-engineered bone regeneration using mesenchymal stem cells and platelet-rich plasma: from basic research to clinical case study

Translational research involves application of basic scientific discoveries into clinically germane findings and, simultaneously, the generation of scientific questions based on clinical observations. At first, as basic research we investigated tissue-engineered bone regeneration using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) in a dog mandible model. We also confirmed the correlation between osseointegration in dental implants and the injectable bone. Bone defects made with a trephine bar were implanted with graft materials as follows: PRP, dog MSCs (dMSCs) and PRP, autogenous particulate cancellous bone and marrow (PCBM), and control (defect only). Two months later, dental implants were installed. According to the histological and histomorphometric observations at 2 months after implants, the amount of bone-implant contact at the bone-implant interface was significantly different between the PRP, PCBM, dMSCs/ PRP, native bone, and control groups. Significant differences were also found between the dMSCs/PRP, native bone, and control groups in bone density. These findings indicate that the use of a mixture of dMSCs/ PRP will provide good results in implant treatment compared with that achieved by autogenous PCBM. We then applied this injectable tissue-engineered bone to onlay plasty in the posterior maxilla or mandible in three human patients. Injectable tissue-engineered bone was grafted and, simultaneously, 2-3 threaded titanium implants were inserted into the defect area. The results of this investigation indicated that injectable tissue-engineered bone used for the plasty area with simultaneous implant placement provided stable and predictable results in terms of implant success. We regenerated bone with minimal invasiveness and good plasticity, which could provide a clinical alternative to autogenous bone grafts. This might be a good case of translational research from basic research to clinical application.     

Bone matrix and marrow versus cancellous bone in rabbit radial defects

Implants of demineralized bone matrix induce new bone formation. In order to estimate the possible clinical usefulness of this phenomenon, autologous cancellous bone grafts were compared with composite grafts of bone matrix and marrow. Cancellous bone from the tuber ischii of the rabbit was transplanted to a preformed radial defect in the same animal. On the opposite side, a similar defect was filled with a mixture of either allogenous or autogenous bone-matrix particles and autogenous bone marrow. After 25 days, calcium 45 was injected intravenously. Three days later the animals were killed. Standardized segments of the rabbit's forearms, containing the middle of the defect, were cut out, ashed, and analyzed for 45Ca activity. No side difference in 45Ca deposition was found. The callus ash weight of the allogenous matrix-transplanted side was approximately 60% of that of the cancellous bone side. This side difference of ash weights corresponds to the estimated initial mineral content of the cancellous graft. Nontransplanted defects had very low ash weight and 45Ca activity. Thus, in the rabbit, composite grafts of bone matrix and marrow produce a bone yield comparable to that of cancellous bone.     

Canine cortical bone autograft remodeling in two simultaneous skeletal sites

The morphological and physical aspects of cortical bone autografts implanted in dogs for 1-9 months in two differently located skeletal defects are reported with a twofold aim: to provide a reference system for further comparison with various allografts and to delineate a general pattern of cortical bone graft healing. A 3-cm osteoperiosteal gap was created in the diaphyseal segment of the ulna and fibula of mature dogs. The grafts, freed from periosteum and bone marrow, were then inverted and replaced for the autografts in the left limb bone without internal fixation or external splints. On the right side, different allografts were tested. A group of three animals also had an unfilled segmental resection on the right as control. Dogs were observed for 1, 2, 3, 6, and 9 months and were able to bear weight within 3 days. Twenty-eight ulnae and 27 fibulae were available for this autograft study. Fluorochromes were injected at mid-term and at the end of the observation. All the grafts were assessed morphologically by cross-section microradiographs and ultraviolet light microscopy, and a morphometric analysis for porosity and fluorescence was done. To evaluate the physical aspects of graft healing, the recovered ulnar autografts, when available, were submitted to photon absorptiometry and to torsional loading. Morphologically, resorption was found to invade the cortical bone graft transversely through radial tunnels, and in addition to the host-bone-graft junction, the entire transplant surface provided another way for revascularization. The highest porosity level was achieved 2 months after surgery for both ulna and fibula, while new bone formation, as assessed by fluorochromes, was most important at 3 months. At 9 months, porosity remained above the normal range as determined in a set of five nongrafted dogs. While the lack of correlation for porosity between the two grafts suggests that local factors are more important in graft resorption, the observed correlation for fluorescence indicates that new bone deposition is more dependent upon skeletal metabolic activity. Within each graft, porosity and new bone formation were not well correlated. In the ulna, the bone mineral content (BMC) reflected the graft volumetric variations during the remodeling, with the lowest mean value at 3 months. For each graft, BMC was well correlated with the torsional stiffness. When torsionally loaded, the maximal tangential shear stress at failure of the graft was negatively related to its cortical porosity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accelerated Healing of Dacron Grafts Seeded by Preclotting with Autologous Bone Marrow Blood

> Studies have suggested that bone marrow-derived cells in the circulation may have the capacity and potential to endothelialize and heal vascular graft surfaces. We have investigated whether accelerated endothelialization could be achieved for Dacron grafts seeded by preclotting with bone marrow blood (BMB). Five 8 mm x 6 cm Dacron grafts seeded and preclotted with BMB and four controls preclotted with peripheral blood were implanted in the descending thoracic aorta (DTA) of mongrel dogs for 2 and 4 weeks. Two additional BMB DTA grafts were studied for 3 months. Five pairs of BMB and control grafts (4 mm x 6 cm) were bilaterally implanted into the carotids of dogs for 1 week and five pairs for 4 weeks. All grafts remained patent. BMB seeding/preclotting was a simple, effective method to accelerate early graft endothelialization without increasing thrombogenicity. Further studies are needed before clinical application can be recommended.     

Comparison of processed bovine internal mammary arteries and autologous veins as arterial femoral substitutes in dogs: blood compatibility and pathological characteristics

This study was undertaken to compare the chemically processed internal mammary artery (BIMA) and the autologous femoral vein as arterial grafts. The BIMA prosthesis was implanted as a left femoral artery bypass and the femoral vein as a right femoral artery bypass graft in 27 dogs. In groups of three dogs the grafts were implanted for predetermined durations: 4, 24 and 48 hours (short term), 1, 2 and 4 weeks (medium term) and 3, 6 and 9 months (long term). All autologous veins were patent when the dogs were killed. The patency rates of the BIMA grafts were 100% in the short-term group, 67% in the medium-term group and 29% in the long-term group. The deposition of labelled fibrinogen and platelets on flow surfaces, the structural preservation of the wall of the BIMA prosthesis and accumulation of thrombi during the period of implantation were studied.     

Biology of bone grafts.

The terminology of bone transplantation is complicated by the various histologic types of bone grafts. A graft moved from one site to another within the same individual is an autograft; the corresponding adjective is autologous or autogenous. An allograft (adjective, allogeneic) is tissue transferred between two genetically different individuals of the same species. A xenograft (adjective, xenogeneic) is tissue from one species implanted into a member of a different species.     

A study of autologous cancellous bone particles in long bone discontinuity defects

Long bone discontinuity defects in dogs were restored by particulate autologous cancellous bone grafts and a Dacron-urethane mesh implant. In six months the discontinuity defects were filled by new bone formation, which was analyzed histologically, radiographically, and by densitometric methods. The postulated mechanism is a field phenomenon of bone induction. The discontinuity defects remained unrestored in the control groups without bone graft material. In the dogs killed at three months incorporation of the graft material was incomplete in the central area of the defect.     

Experimental application of calcium phosphate granulate for the substitution of conventional bone transplants (author's transl)]

Autologous spongiosa, a calcium phosphate ceramic and Kiel bone chips were implanted in the tibiae of dogs and compared with respect to tissue compatibility and osteogenetic effect. After the ceramic implants and the autologous spongiosa had been left in the tibial fat marrow for six weeks, bone tissue and bone marrow had formed to the same extent around both materials. Their stimulating effect on osteogenesis was comparable. In contrast to the ceramic material, together with which they had been implanted in active bone marrow, the Kiel bone chips were surrounded by fibrous tissue in addition to bone tissue. All of the three types of implant proved to be tissue compatible. On the whole, the calcium phosphate ceramic was found to be equal to autologous and superior to heterologous spongiosa from a biological point of view. In technical terms the ceramic implant was superior also to the autologous graft.     

The influence of hydroxyapatite granules on the healing of a segmental defect filled with autologous bone marrow.

Hydroxyapatite(HA) ceramics are frequently used as a bone graft substitutes for the filling of bony defects. The addition of autologous bone marrow to HA ceramics does improve defect healing. There is conflicting evidence in the literature whether autologous bone marrow transplantation alone is as effective as the combination of HA ceramics and bone marrow combined. It was the purpose of this study to identify the role of additional HA ceramic granules on the healing of a sheep tibia segmental defect filled with autologous bone marrow. After permission of the local animal rights committee was obtained, a 3 cm segmental defect in the midshaft of 31 adult sheep was stabilized with an unreamed tibia nail. The animals were divided into 4 groups according to the mode of defect filling: HA plus autologous bone marrow (HA + MAR) (n = 8), autologous bone marrow (MAR) (n = 9), empty defect (DEF) (n = 6), cancellous bone graft (CAN) (n = 8). After three months follow up animals were sacrificed and analysed for the key parameters of union and maximum torque at failure. One nonunion was present in each of the HA + MAR, MAR, and CAN groups. Four of the six animals in the DEF group developed a nonunion. Maximum torque at failure was reported as percentage of the intact contralateral tibia: HA + MAR 39% +/- 24%, MAR 26% +/- 17%, DEF 22% +/- 13%, CAN 41% +/- 20%. The difference between the groups was statistically significant, but appeared to be relevant. We conclude from our data, that HA ceramics do improve healing of a segmental defect in the sheep tibia filled with autologous bone marrow. The results of this combination are comparable to cancellous autograft.     

Autogeneic bone marrow and porous biphasic calcium phosphate ceramic for segmental bone defects in the canine ulna.

The purpose of this study was to evaluate a porous biphasic hydroxyapatite-calcium phosphate ceramic as a modifier and extender of an autogeneic marrow graft for filling a 2.5-cm segmental bony defect. Twenty adult mongrel dogs were surgically treated to create diaphyseal defects in the left ulnae. The defects were (1) filled with autogeneic bone marrow mixed with granular hydroxyapatite-tricalcium phosphate ceramic (granular ceramic); (2) grafted with a solid block of ceramic soaked in autogeneic bone marrow (block ceramic); (3) received no graft (no implant); or (4) were grafted with autogeneic bone marrow alone (bone marrow). All animals were followed clinically and roentgenographically for 24 weeks and then killed. Repair of diaphyseal defects with the block ceramic led to three solid unions and three fibrous unions; with the granular ceramic implants and marrow, the defects of five dogs formed solid unions, and one progressed to a fibrous union. Defects in all five dogs grafted with autogeneic bone marrow united. The three dogs with no implant formed nonunions. Histology showed normal marrow and only a light immune reaction. Complete bridging of the defect in the dogs treated with the granular ceramic occurred significantly earlier than bridging in the dogs grafted with bone marrow alone. Histomorphometry, performed on the block ceramic implants indicated active resorption of ceramic. Clinically, addition of ceramic to a marrow graft improved the handling characteristics of the graft material and accelerated healing according to roentgenographic evaluation.