Shock wave application enhances pertussis toxin protein-sensitive bone formation of segmental femoral defect in rats.

Extracorporeal shock waves (ESWs) elicit a dose-dependent effect on the healing of segmental femoral defects in rats. After ESW treatment, the segmental defect underwent progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Along with the intensive bone formation, there was a persistent increase in TGF-beta1 and BMP-2 expression. Pretreatment with pertussis toxin reduced ESW-promoted callus formation and gap healing, which presumably suggests that Gi proteins mediate osteogenic signaling. INTRODUCTION: Extracorporeal shock waves (ESWs) have previously been used to promote bone repair. In our previous report, we found that ESWs promoted osteogenic differentiation of mesenchymal cells through membrane perturbation and activation of Ras protein. In this report, we show that ESWs elicit a dose-dependent effect on the healing of segmental defects and that Gi proteins play an important role in mediating ESW stimulation. MATERIALS AND METHODS: Rats with segmental femoral defects were subjected to ESW treatment at different energy flux densities (EFD) and impulses. Bone mass (mineral density and calcium content), osteogenic activities (bone alkaline phosphatase activity and osteocalcin content), and immunohistochemistry were assessed. RESULTS: An optimal ESW energy (500 impulses at 0.16 mJ/mm2 EFD) stimulated complete bone healing without complications. ESW-augmented healing was characterized by significant increases (p < 0.01) in callus size, bone mineral density, and bone tissue formation. With exposure to ESW, alkaline phosphatase activity and osteocalcin production in calluses were found to be significantly enhanced (p < 0.05). After ESW treatment, the histological changes we noted included progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Intensive bone formation was associated with a persistent increase in transforming growth factor-beta 1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) expression, suggesting both growth factors were active in ESW-promoted bone formation. We also found that pertussis toxin, an inhibitor of membrane-bound Gi proteins, significantly reduced (p < 0.01) ESW promotion of callus formation and fracture healing. CONCLUSION: ESW treatments enhanced bone formation and the healing of segmental femoral defects in rats. It also seems likely that TGF-beta1 and BMP-2 are important osteogenic factors for ESW promotion of fracture healing, presumably through Gi protein-mediated osteogenic signaling.     

Extracorporeal shock waves enhance normal fibroblast proliferation in vitro and activate mRNA expression for TGF-��1 and for collagen types I and III

Background and purpose Extracorporeal shock waves (ESWs) are used to good effect in the treatment of soft tissue injuries, but the underlying mechanisms are still unknown. We therefore determined the effects of ESWs on normal fibroblasts in vitro, in order to assess treatment-induced cell response.Methods A normal human fibroblast cell line (NHDF-12519) was treated with ESWs generated by a piezoelectric device (Piezoson 100; Richard Wolfe) using different protocols of impulses (300, 1,000, or 2,000 shots) and energy (0.11 or 0.22 mJ/mm²). Untreated controls and treated cells were cultivated for 12 days following a single shock-wave treatment. Viability, growth rate, and expression of mRNA for TGFβ -1 and collagen types I and III were evaluated at days 3, 6, 9, and 12.Results 1 hour after shock-wave treatment, cell viability showed a decrease related mainly to impulse numbers applied. Fibroblasts treated with energy of 0.22 mJ/mm² subsequently showed an increase in proliferation from day 6 to day 9 that was higher than in untreated controls, without interference with the normal cell kinetic profile. mRNA expression was also higher in treated fibroblasts than in untreated controls for TGFβ -1 on day 6 and day 9, for collagen type I on day 6, and for collagen type III on day 9.Interpretation These in vitro data confirm that the main factors involved in the repair process of connective tissues are activated by ESWs. The study gives the rationale for, and may provide schedules for, ESW treatment of tendonopathies.     

Cambium cell stimulation from surgical release of the periosteum.

An autograft of periosteal tissue containing cambium cells has potential to become chondrogenic or osteogenic depending on the regeneration repair strategies. The potential number of harvestable cambium cells diminishes with age. Other factors may be associated with a reduction in the number or variable yields of cambium cells including harvest technique, harvest site location, and the time interval from harvest to implantation. Attempts to increase the number of cambium cells have included improvements in harvesting and handling technique, and expansion of the cells in tissue culture. An "in situ" stimulation and proliferation technique would offer the potential for increasing the number of cambium cells in a cost-effective manner for transplantation without the need for expansion in tissue culture.The hypothesis tested was that surgical release of the periosteum and its deep inner underlying cambium layer by sharply incising through the superficial periosteal fibrous layer down to and scoring the cortical bone surface would increase the number of cambium cells that could be harvested at a later time period. Two techniques for periosteal release were used to stimulate a proliferation of the underlying cambium layer and increase the cambium cells for harvest in skeletally mature goats: (1) sharply scoring all four-sides of the tissue test site perimeter, and (2) sharply scoring only two sides of the tissue test site.The two-sided and four-sided release scoring of the periosteum induced stimulatory responses in the number of cambium cells. In addition, a marked increase in mRNA expression for BMP-2 (p<0.001) was observed within 24 h and remained elevated over baseline values for up to 96 h after this stimulation to the cambium layer.     

Hydraulic elevation of the periosteum: a novel technique for periosteal harvest.

Periosteum has been promoted as a potential substrate for tissue engineering. Its principal virtues are that it has a source of pluripotential mesenchymal cells and chondrogenic growth factors located in the cambium layer, and it can serve as a template for directional evolution of neo-tissue. The clinical use and in vitro study of periosteum-derived neo-tissue has been limited by the level of surgical skill required for harvest. Precise surgical technique, task-specific experience, adequate volume of procedures, and general surgical expertise are required for optimal harvest using the traditional periosteal elevator method. This report describes an easily mastered technique that preserves viability while providing the harvest of relatively large amounts of periosteum. Skeletally mature New Zealand white rabbits (11 males/20 tibias; 4 females/8 tibias; approximate weight 3.5 kg) and one Yucatan miniature pig were used for harvest of periosteum from the tibia using the traditional periosteal elevator and the developed hydraulic elevation approach. Histologic examination of the periosteal explants obtained by the developed method showed preservation of the cambium layer containing the progenitor cells necessary for the generation of neo-cartilage. This technique provides a simple method of harvesting large segments (>5 cm x 1 cm) of periosteum in a single procedure and may facilitate better exploitation of periosteum in tissue engineering.     

Periosteum responds to dynamic fluid pressure by proliferating in vitro.

Periosteum provides a source of undifferentiated chondrocyte precursor cells for fracture healing that can also be used for cartilage repair. The quantity of cartilage that can be produced, which is a determining factor in fracture healing and cartilage repair, is related to the number of available stem cells in the cambium layer. Cartilage formation during both of these processes is enhanced by motion of the fracture or joint in which periosteum has been transplanted. The effect of dynamic fluid pressure on cell proliferation in periosteal tissue cultures was determined in 452 explants from 60 immature (2-month-old) New Zealand White rabbits. The explants were cultured in agarose suspension for 1-14 days. One group was subjected to cyclic hydrostatic pressure, which is referred to as dynamic fluid pressure, at 13 kPa and a frequency of 0.3 Hz. Control explants were cultured in similar chambers without application of pressure. DNA synthesis ([3H]thymidine uptake) and total DNA were measured. The temporal pattern and distribution of cell proliferation in periosteum were evaluated with autoradiography and immunostaining with proliferating cell nuclear antigen. Dynamic fluid pressure increased proliferation of periosteal cells significantly, as indicated by a significant increase in [3H]thymidine uptake at all time points and a higher amount of total DNA compared with control values. On day 3, when DNA synthesis reached a peak in periosteal explants, [3H]thymidine uptake was 97,000+/-5,700 dpm/microg DNA in the group exposed to dynamic fluid pressure and 46,000+/-6,000 dpm/microg in the controls (p < 0.001). Aphidicolin, which blocks DNA polymerase alpha, inhibited [3H]thymidine uptake in a dose-dependent manner in the group subjected to dynamic fluid pressure as well as in the positive control (treated with 10 ng/ml of transforming growth factor-beta1) and negative control (no added growth factors) groups, confirming that [3H]thymidine measurements represent proliferation and dynamic fluid pressure stimulates DNA synthesis. Total DNA was also significantly higher in the group exposed to dynamic fluid pressure (5,700+/-720 ng/mg wet weight) than in the controls (3,700+/-630) on day 3 (p < 0.01). Autoradiographs with [3H]thymidine revealed that one or two cell cycles of proliferation took place in the fibrous layer prior to proliferation in the cambium layer (where chondrocyte precursors reside). Proliferating cell nuclear antigen immunophotomicrographs confirmed the increased proliferative activity due to dynamic fluid pressure. These findings suggest either a paracrine signaling mechanism between the cells in these two layers of the periosteum or recruitment/migration of proliferating cells from the fibrous to the cambium layer. On the basis of the data presented in this study, we postulate that cells in the fibrous layer respond initially to mechanical stimulation by releasing growth factors that induce undifferentiated cells in the cambium layer to divide and differentiate into chondrocytes. These data indicate that cell proliferation in the early stages of chondrogenesis is stimulated by mechanical factors. These findings are important because they provide a possible explanation for the increase in cartilage repair tissue seen in joints subjected to continuous passive motion. The model of in vitro periosteal chondrogenesis under dynamic fluid pressure is valuable for studying the mechanisms by which mechanical factors might be involved in the formation of cartilage in the early fracture callus and during cartilage repair.     

Differential phenotypic characteristics of heterogeneous cell population in the rabbit periosteum

Background Periosteum and periosteum-derived progenitor cells have demonstrated the potential for stimulative applications in repair of various musculoskeletal tissues. It has been found that the periosteum contains mesenchymal progenitor cells that are capable of differentiating into either osteoblasts or chondrocytes, depending on the culture conditions. Anatomically, the periosteum is a heterogeneous multilayered membrane, consisting of an outer fibrous and an inner cambium layer. The present study was designed to elucidate the phenotypic characteristics of fibrous and cambium layer cells in vitro.

Methods Using a sequential enzymatic digestion method, fibrous and cambium layer cells were harvested separately from periosteum-bone explants of the proximal tibia of 6-month-old New Zealand White rabbits.

Results We found that the cells from each layer showed distinct phenotypic characteristics in a primary monolayer culture system. Specifically, the cambium cells demonstrated higher osteogenic characteristics (higher alkaline phosphatase and osteocalcin levels) than the fibrous cells. However, these differences diminished with time in vitro.

Interpretation Our findings suggest that the periosteum has phenotypically distinct heterogeneous cell populations. Care must be taken in order to identify and distinguish the intrinsic phenotypes of the heterogeneous periosteum-derived cell types in vitro.     

Extracorporeal shock waves enhance normal fibroblast proliferation in vitro and activate mRNA expression for TGF-β1 and for collagen types I and III

Background and purpose Extracorporeal shock waves (ESWs) are used to good effect in the treatment of soft tissue injuries, but the underlying mechanisms are still unknown. We therefore determined the effects of ESWs on normal fibroblasts in vitro, in order to assess treatment-induced cell response.

Methods A normal human fibroblast cell line (NHDF-12519) was treated with ESWs generated by a piezoelectric device (Piezoson 100; Richard Wolfe) using different protocols of impulses (300, 1,000, or 2,000 shots) and energy (0.11 or 0.22 mJ/mm²). Untreated controls and treated cells were cultivated for 12 days following a single shock-wave treatment. Viability, growth rate, and expression of mRNA for TGFβ -1 and collagen types I and III were evaluated at days 3, 6, 9, and 12.

Results 1 hour after shock-wave treatment, cell viability showed a decrease related mainly to impulse numbers applied. Fibroblasts treated with energy of 0.22 mJ/mm² subsequently showed an increase in proliferation from day 6 to day 9 that was higher than in untreated controls, without interference with the normal cell kinetic profile. mRNA expression was also higher in treated fibroblasts than in untreated controls for TGFβ -1 on day 6 and day 9, for collagen type I on day 6, and for collagen type III on day 9.

Interpretation These in vitro data confirm that the main factors involved in the repair process of connective tissues are activated by ESWs. The study gives the rationale for, and may provide schedules for, ESW treatment of tendonopathies.     

Differential phenotypic characteristics of heterogeneous cell population in the rabbit periosteum

BACKGROUND: Periosteum and periosteum-derived progenitor cells have demonstrated the potential for stimulative applications in repair of various musculoskeletal tissues. It has been found that the periosteum contains mesenchymal progenitor cells that are capable of differentiating into either osteoblasts or chondrocytes, depending on the culture conditions. Anatomically, the periosteum is a heterogeneous multilayered membrane, consisting of an outer fibrous and an inner cambium layer. The present study was designed to elucidate the phenotypic characteristics of fibrous and cambium layer cells in vitro. METHODS: Using a sequential enzymatic digestion method, fibrous and cambium layer cells were harvested separately from periosteum-bone explants of the proximal tibia of 6-month-old New Zealand White rabbits. RESULTS: We found that the cells from each layer showed distinct phenotypic characteristics in a primary monolayer culture system. Specifically, the cambium cells demonstrated higher osteogenic characteristics (higher alkaline phosphatase and osteocalcin levels) than the fibrous cells. However, these differences diminished with time in vitro. INTERPRETATION: Our findings suggest that the periosteum has phenotypically distinct heterogeneous cell populations. Care must be taken in order to identify and distinguish the intrinsic phenotypes of the heterogeneous periosteum-derived cell types in vitro.     

Behaviour of cancellous bone graft with and without periosteal isolation in striated muscle. An experimental study

The capacity of the periosteum to inhibit resorption of cancellous bone grafts into muscle was investigated in 34 four- to six-week-old rabbits. In 17 experiments the periosteum was wrapped around the grafts with the cambium layer facing the bone, and in seven experiments with the cambium layer facing the muscle. In the control group of 10 experiments there was no periosteal wrapping around the bone grafts. In Series 1 with the cambium layer of the periosteum facing the bone, after 20 weeks a tubular bone with Haversian system and bone marrow was seen. The transplants were surrounded by normal-looking periosteum. Bone formation from the periosteum occurred through enchondral ossification. Inductive bone growth was observed from the cancellous graft. In Series 2 with the cambium layer facing the surrounding muscle tissue, after 20 weeks two laminar bone blocks with periosteum in between and surrounding each block was observed. In the control series without periosteal covering, after 20 weeks only fibrous tissue remained in the transplantation site. It is obvious that periosteal isolation of cancellous bone grafts inhibits their resorption when transplanted into muscle in young animals.     

Comparison of bioengineered human bone construct from four sources of osteogenic cells

Osteoprogenitor cells have been reported to be present in periosteum, cancellous and cortical bone, and bone marrow; but no attempt to identify the best cell source for bone tissue engineering has yet been reported. In this study, we aimed to investigate the growth and differentiation pattern of cells derived from these four sources in terms of cell doubling time and expression of osteoblast-specific markers in both monolayer cells and three-dimensional cell constructs in vitro. In parallel, human plasma derived-fibrin was evaluated for use as biomaterial when forming three-dimensional bone constructs. Our findings showed osteoprogenitor cells derived from periosteum to be most proliferative followed by cortical bone, cancellous bone, and then bone marrow aspirate. Bone-forming activity was observed in constructs formed with cells derived from periosteum, whereas calcium deposition was seen throughout the constructs formed with cells derived from cancellous and cortical bones. Although no mineralization activity was seen in constructs formed with osteoprogenitor cells derived from bone marrow, well-organized lacunae as would appear in the early phase of bone reconstruction were noted. Scanning electron microscopy evaluation showed cell proliferation throughout the fibrin matrix, suggesting the possible application of human fibrin as the bioengineered tissue scaffold at non-load-bearing sites.     

Extracorporeal shock waves promote healing of collagenase-induced Achilles tendinitis and increase TGF-beta1 and IGF-I expression.

Extracorporeal shock waves (ESW) have recently been used in resolving tendinitis. However, mechanisms by which ESW promote tendon repair is not fully understood. In this study, we reported that an optimal ESW treatment promoted healing of Achilles tendintis by inducing TGF-beta1 and IGF-I. Rats with the collagenease-induced Achilles tendinitis were given a single ESW treatment (0.16 mJ/mm(2) energy flux density) with 0, 200, 500 and 1000 impulses. Achilles tendons were subjected to biomechanical (load to failure and stiffness), biochemical properties (DNA, glycosaminoglycan and hydroxyproline content) and histological assessment. ESW with 200 impulses restored biomechanical and biochemical characteristics of healing tendons 12 weeks after treatment. However, ESW treatments with 500 and 1000 impulses elicited inhibitory effects on tendinitis repair. Histological observation demonstrated that ESW treatment resolved edema, swelling, and inflammatory cell infiltration in injured tendons. Lesion site underwent intensive tenocyte proliferation, neovascularization and progressive tendon tissue regeneration. Tenocytes at the hypertrophied cellular tissue and newly developed tendon tissue expressed strong proliferating cell nuclear antigen (PCNA) after ESW treatment, suggesting that physical ESW could increase the mitogenic responses of tendons. Moreover, the proliferation of tenocytes adjunct to hypertrophied cell aggregate and newly formed tendon tissue coincided with intensive TGF-beta1 and IGF-I expression. Increasing TGF-beta1 expression was noted in the early stage of tendon repair, and elevated IGF-I expression was persisted throughout the healing period. Together, low-energy shock wave effectively promoted tendon healing. TGF-beta1 and IGF-I played important roles in mediating ESW-stimulated cell proliferation and tissue regeneration of tendon.     

The periosteum. Part 1: Anatomy, histology and molecular biology

The periosteum is a thin layer of connective tissue that covers the outer surface of a bone in all places except at joints (which are protected by articular cartilage). As opposed to bone itself, it has nociceptive nerve endings, making it very sensitive to manipulation. It also provides nourishment in the form of blood supply to the bone. The periosteum is connected to the bone by strong collagenous fibres called Sharpey's fibres, which extend to the outer circumferential and interstitial lamellae of bone. The periosteum consists of an outer "fibrous layer" and inner "cambium layer". The fibrous layer contains fibroblasts while the cambium layer contains progenitor cells which develop into osteoblasts that are responsible for increasing bone width. After a bone fracture the progenitor cells develop into osteoblasts and chondroblasts which are essential to the healing process. This review discusses the anatomy, histology and molecular biology of the periosteum in detail.     

Localization of chondrocyte precursors in periosteum

OBJECTIVE: Periosteal chondrogenesis is relevant to cartilage repair and fracture healing. Periosteum contains two distinct layers: a thick, outer fibrous layer and a thin, inner cambium layer which is adjacent to the bone. Specific chondrocyte precursors are known to exist in periosteum but have not yet been identified. In this study, the location of the chondrocyte precursors in periosteum was determined. METHOD: One hundred and twenty periosteal explants from 30 2-month-old NZ rabbits were cultured for up to 42 days. Histomorphological changes and spatio-temporal localization of Col. II mRNA and protein were analysed. RESULTS: On day 7, chondrocyte differentiation appeared in the most juxtaosseous region in the cambium layer. Col. II mRNA and protein were also evident in the same region. By day 14, chondrocyte differentiation progressed further into the juxtaosseous cambium layer, as did Col. II mRNA and protein. With growth of the neocartilage, the cambium layer gradually diminished to the extent that by 21-28 days it was no longer evident. Cartilage growth was significant and followed an appositional pattern, growing away from the fibrous layer. The fibrous layer remained essentially unchanged from 0-42 days, without evidence of hypertrophy or atrophy. Col. II mRNA expression was never seen in the fibrous layer. CONCLUSION: From these data, three conclusions can be drawn concerning chondrogenesis from periosteum: (1) the chondrocyte precursors are located in the cambium layer of periosteum; (2) chondrogenesis commences in the juxtaosseous area in the cambium layer and progresses from the juxtaosseous region to the juxtafibrous region of the cambium layer; (3) neocartilage growth is appositional, which displaces the fibrous layer away from the cartilage already formed, as new cartilage is formed between these two layers. These findings suggest that the least differentiated (stem or reserve) cells are located in the cambium layer furthest from the bone. CLINICAL RELEVANCE: These findings show that the chondrocyte precursors are located in the cambium layer of periosteum. Preservation of this layer is essential for chondrogenesis. As neocartilage growth is appositional, away from the fibrous layer, it can be expected that the new cartilage deposited in and adjacent to a periosteal graft would be expected to be located on the side of the cambium layer, rather than on the side of the fibrous layer of the graft.     

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA‐segmental defect rat model

A layer of cells (the “biomembrane”) has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic‐impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non‐unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1198–1212, 2012     

Extracorporeal shock wave treatment modulates skin fibroblast recruitment and leukocyte infiltration for enhancing extended skin-flap survival

Extracorporeal shock wave (ESW) treatment has a positive effect of rescuing ischemic skin flaps. This study assessed whether ESW treatment rescues the compromised flap tissue by suppressing the apoptosis of ischemic tissue and recruiting tissue remodeling. We used a random-pattern extended dorsal–skin-flap (10 × 3 cm) rodent model. Thirty-six male Sprague–Dawley rats were divided into three groups. Group I, the control group, received no treatment. Group II received one session of ESW treatment (500 impulses at 0.15 mJ/mm²) immediately after surgery. Group III received two sessions of ESW treatment, immediately and the day after the surgery. Results indicated that the necrotic area in the flaps in group II was significantly smaller than that of the flaps in group I ( p <0.01). Transferase dUTP-nick end labeling (TUNEL) analysis revealed a significant decrease in the number of apoptotic cells in group II. Hydrogen peroxide (H₂O₂) expression in circulation blood was significantly decreased in group II on the day after ESW treatment. Immunohistochemical staining indicated that compared with no treatment, ESW treatment could substantially increase proliferating cell nuclear antigen (PCNA), endothelial nitric oxide synthase, and prolyl 4-hydroxylase (rPH) expression, reduce CD45 expression, and suppress 8-hydroxyguanosine (8-OG) expression in the ischemic zone of the flap tissue. In conclusion, ESW treatment administered at an optimal dosage exerts a positive effect of rescuing ischemic extended skin flaps. The mechanisms of action of ESWs involve modulation of oxygen radicals, attenuation of leukocyte infiltration, decrease in tissue apoptosis, and recruitment of skin fibroblasts, which results in increased flap tissue survival.     

Vascularized bone graft from the supracondylar region of the femur

Free vascularized thin corticoperiosteal grafts and small periosteal bone grafts harvested from the supracondylar region of the femur are described. These grafts are nourished from the articular branch of the descending genicular artery and vein. Unlike currently used vascularized bone grafts, this graft can be successfully harvested with disturbing the vascularity. Thin corticoperiosteal grafts consist of periosteum with a thin layer of outer cortical bone and include the cambium layer, which has a better osteogenic capacity. This graft is elastic and readily conforms to the recipient bed configuration. Thin corticoperiosteal grafts were used for fracture nonunion of the long bone with smaller bone defect and to treat forty‐six patients with avascular necrosis of the body of the talus, scaphoid, and lunate bone. © 2009 Wiley‐Liss, Inc. Microsurgery 2009.     

Osteogenesis induced by extracorporeal shockwave in treatment of delayed osteotendinous junction healing

Healing at the osteotendinous junction (OTJ) is challenging in orthopedic surgery. The present study aimed to test extracorporeal shockwave (ESW) in treatment of a delayed OTJ healing. Twenty‐eight rabbits were used for establishing a delayed healing (DH) model at patella‐patellar‐tendon (PPT) complex after partial patellectomy for 4 weeks and then were divided into DH and ESW groups. In the ESW group, a single ESW treatment was given at postoperative week 6 to the PPT healing complex. The samples were harvested at week 8 and 12 for radiographic and histological evaluations with seven samples for each group at each time point. Micro‐CT results showed that new bone volume was 1.18 ± 0.61 mm³ in the ESW group with no measurable new bone in the DH group at postoperative week 8. Scar tissue formed at the OTJ healing interface of the DH group, whereas ESW triggered high expression of VEGF in hypertrophic chondrocytes at week 8 and regeneration of the fibrocartilage zone at week 12 postoperatively. The accelerated osteogenesis could be explained by acceleration of endochondral ossification. In conclusion, ESW was able to induce osteogenesis at OTJ with delayed healing with enhanced endochondral ossification process and regeneration of fibrocartilage zone. These findings formed a scientific basis to potential clinical application of ESW for treatment of delayed OTJ healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:70–76, 2010     

体外震波治疗对糖尿病大鼠慢性创面愈合及组织学影响

目的体外震波(extracorporeal shock wave,ESW)可促进血管新生和组织修复,通过观察低能量ESW治疗糖尿病大鼠慢性创面的效果,探讨其促进糖尿病慢性创面愈合的机制。方法 6～8周龄雄性SD大鼠96只,体重(220±20)g,随机分为3组(糖尿病对照组、ESW治疗组及正常对照组),每组32只。取糖尿病对照组、ESW治疗组大鼠,腹腔注射链脲佐菌素(60 mg/kg)制备糖尿病大鼠模型后,于背部制备1个直径1.8 cm的圆形皮肤全层创面,建立糖尿病大鼠慢性创面模型。正常对照组大鼠同法制备创面。创面制备后1 d ESW治疗组采用0.11 mJ/mm2、1.5 Hz的ESW对创面干预500脉冲;糖尿病对照组及正常对照组创面未行ESW治疗。观察创面愈合情况;于3、7、14 d取创面组织行HE及Masson染色,观察创面组织学变化;行CD31和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)免疫组织化学染色,观察创面细胞增殖及血管化程度。结果糖尿病对照组创面闭合率低于正常对照组,创面闭合时间延长(P<0.05),治疗后3、7、14 d,创面组织炎性细胞浸润明显,胶原纤维相对面积密度、创面微血管密度、PCNA阳性细胞相对密度均低于正常对照组(P<0.05)。ESW治疗组与糖尿病对照组相比,创面愈合时间缩短,创面闭合率提高(P<0.05),各时间点炎性细胞计数减少,肉芽组织生长良好,创面内胶原纤维相对面积密度、微血管密度和PCNA阳性细胞相对密度增加,差异有统计学意义(P<0.05);ESW治疗组与正常对照组相比,微血管密度和PCNA阳性细胞相对密度差异均无统计学意义(P>0.05)。结论低能量ESW治疗可以抑制糖尿病大鼠慢性创面内局部炎性反应,提高创面内修复细胞增殖能力,增加微血管形成和胶原沉积,使肉芽组织形成增加,最终促进创面愈合。     

新西兰大白兔骨膜下组织工程骨异位成骨的实验研究

目的：利用新西兰大白兔骨髓基质干细胞构建组织工程骨,研究其在股骨骨膜下异位成骨的可行性。方法：选取3月龄的清洁级雌性新西兰大白兔,体重3kg,取其骨髓基质干细胞诱导为成骨干细胞,扩增后接种到β-磷酸三钙生物陶瓷颗粒中,构建的组织工程骨种植到股骨骨膜下,3个月后实验动物血管内灌注凝胶墨汁,通过光学显微镜直接检测组织工程骨的血供和成骨结果。结果：16个标本中的12个植入的组织工程骨颗粒均良好固定在骨膜下并被骨膜包围,组织工程骨中有大量血管和新生骨形成,骨组织结构相对紊乱,不同于正常骨组织结构排列规则,血管分布均匀。4例取材时发现植入物游离于骨膜外,大部分材料被吸收,残留植入物体积明显小于骨膜下成骨,未见明显的骨组织形成,血供情况欠佳。股骨骨膜下组织工程骨颗粒80%贴附牢固,成骨良好,新骨内有大量血管长入。结论：组织工程骨可以在骨膜下获得良好的异位成骨。     

Structural and cellular differences between metaphyseal and diaphyseal periosteum in different aged rats

In both physiological and pathological processes, periosteum plays a determinant role in bone formation and fracture healing. However, no specific report is available so far focusing on the detailed structural and major cellular differences between the periostea covering different bone surface in relation to ageing. The aim of this study is to compare the structural and cellular differences in diaphyseal and metaphyseal periostea in different aged rats using histological and immunohistochemical methods. Four female Lewis rats from each group of juvenile (7 weeks old), mature (7 months old) and aged groups (2 years old) were sacrificed and the right femur of each rat was retrieved, fixed, decalcified and embedded. Five-micrometer thick serial sagittal sections were cut and stained with Hematoxylin and Eosin, Stro-1 (stem cell marker), F4/80 (macrophage marker), TRAP (osteoclast marker) and vWF (endothelial cell marker). One-millimeter lengths of middle diaphyseal and metaphyseal periosteum were selected for observation. The thickness, total cell number and positive cell number for each antibody were measured and compared in each periosteal area and different aged groups. The results were subjected to two-way ANOVA and SNK tests. The results showed that the thickness and cell number in diaphyseal periosteum decreased with age (p<0.001). In comparison with diaphyseal area, the thickness and cell number in metaphyseal periosteum were much higher (p<0.001). There were no significant differences between the juvenile and aged groups in the thickness and cell number in the cambial layer of metaphyseal periosteum (p>0.05). However, the juvenile rats had more Stro1(+), F4/80(+) cells and blood vessels and fewer TRAP(+) cells in different periosteal areas compared with other groups (p<0.001). The aged rats showed much fewer Stro1(+) cells, but more F4/80(+), TRAP(+) cells and blood vessels in the cambial layer of metaphyseal periosteum (p<0.001). In conclusion, structure and cell population of periosteum appear to be both age-related and site-specific. The metaphyseal periosteum of aged rats seems more destructive than diaphyseal part and other age groups. Macrophages in the periosteum may play a dual important role in osteogenesis and osteoclastogenesis.