Porcine enamel matrix derivative enhances trabecular bone regeneration during wound healing of injured rat femur.

To elucidate the effects of enamel matrix derivative (EMD: Emdogain) on bone regeneration in rat femurs after drill-hole injury, defects in bone were filled with either EMD or its carrier, PGA, as control. On postoperative days 4 to 28, dissected femurs were examined by means of various morphological approaches. In both experimental groups, formation of trabecular bone, which was immunostained for bone sialoproteins (BSP), had occurred in the medullary cavities at cylindrical bone defects on Day 7 postoperatively. Cuboidal osteoblasts were clearly observed on these newly-formed BSP-positive bone trabeculae. On Days 7 and 14, many multinucleated giant cells, which strongly expressed cathepsin K, had appeared on these bone trabeculae, indicating active bone remodeling. In these bone trabeculae, Ca and P weight % and Ca/P ratio were similar to those of cortical bone, and there was no significant difference between the PGA- and EMD-applied groups. Bone volume fraction of newly-formed bone trabeculae on Day 7 postoperatively was significantly higher in the EMD-applied group than in the PGA-applied controls. Because of active bone remodeling and the marked decrease of bone volume, on Days 14 and 28 postoperatively, however, there was no longer a significant difference in trabecular bone volume fraction between the experimental groups. Our results suggest that EMD possesses an osteo-promotive effect on bone and medullary regeneration during wound healing of injured long bones.     

SD大鼠骨基质明胶吸附骨髓间充质干细胞修复骨缺损的实验研究

目的:探讨大鼠骨基质明胶吸附骨髓间充质干细胞修复骨缺损的可行性。方法:采用第5代(P5)SD大鼠骨髓间充质干细胞(MSCs),经Hoeschst 33344(sigma)荧光杂料标记后,调配制成的1×106个／ml细胞浓度后与骨基质明胶(BMG)共同培养6 h,然后植入SD大鼠双侧胫骨的实验性骨缺损中(A组),同时作胫骨骨缺损单纯BMG植入(B组),无植入物(C组)两组对照。术后8周处死,取骨缺损区组织进行组织学观察,其中A组行荧光染料标记测定以确认骨缺损区的骨痂是否来源于MSCS。结果:①A组:胫骨骨缺损区可见大量新生不规则骨纤维组织、软骨及纤维骨痂填充,可见骨细胞、骨组织和骨小梁,已形成骨髓腔。②B组:胫骨骨缺损区可见大量纤维组织、少量新生不规则骨纤维组织及骨骼肌组织,伴有多核巨细胞和少量炎性细胞,缺损区边缘带有骨痂组织。③C组:胫骨骨缺损区可见大量纤维组织及骨骼肌组织填充生长,伴有多核巨细胞和少量炎性细胞,缺损区边缘带有少量骨痂组织。荧光染色鉴定确认胫骨骨缺损区的骨痂来源于MSCs。结论:大鼠骨基质明胶吸附骨髓间充质干细胞修复骨缺损具有安全性、可行性及有效性。     

Effect of xenogenic immobilized bone matrix on the course of wound process

The effect of xenogenic demineralized bone matrix as a component of film coating on healing of a full-thickness skin wound was studied. Regenerative processes were more intensive in wounds dressed with a complex film containing demineralized bone matrix than in uncovered wounds and wounds covered with films without bone matrix. Film coating with antibiotics suppressed the development of wound microflora. In vitro experiments demonstrated different effects of demineralized bone matrix on some enzymes in rat skin and liver homogenates. Biological activity of xenogenic demineralized bone matrix as a component of wound coating is shown. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 448–450, April, 2006     

Effect of thyrocalcitonin on the mineral component of regenerating bone during healing of fractures

Changes in the state of the mineral component of bone tissue (hydroxyapatite) during reparative regeneration of bone after administration of thyrocalcitonin (TCT) were studied in experiments on rabbits. Methods of histology, quantitative microroentgenography, and x-ray diffraction were used. TCT has no marked stimulant effect on normal osteogenesis and mineralization of newly formed bone tissue. However, mineralization of the callus follows an optimal course and osteoporosis of the fragments is much less marked than in animals not receiving TCT.Central Institute of Traumatology and Orthopedics, Ministry of Health of the USSR. All-Union Scientific-Research Institute of Antibiotics, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 78, No. 8, pp. 103–105, August, 1974.     

Transplantation of bone marrow mesenchymal stem cells on collagen scaffolds for the functional regeneration of injured rat uterus

Serious injuries of endometrium in women of reproductive age are often followed by uterine scar formation and a lack of functional endometrium predisposing to infertility or miscarriage. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown great promise in clinical applications. In the present study, BM-MSCs loaded onto degradable collagen membranes were constructed. Collagen membranes provided 3-dimmensional architecture for the attachment, growth and migration of rat BM-MSCs and did not impair the expression of the stemness genes. We then investigated the effect of collagen/BM-MSCs constructs in the healing of severe uterine injury in rats (partial full thickness uterine excision). At four weeks after the transplantation of collagen/BM-MSCs constructs, BM-MSCs were mainly located to the basal membrane of regenerative endometrium. The wounded tissue adjacent to collagen/BM-MSCs constructs expressed higher level of bFGF, IGF-1, TGFβ1 and VEGF than the corresponding tissue in rats receiving collagen construct alone or in spontaneous regeneration group. Moreover, the collagen/BM-MSCs system increased proliferative abilities of uterine endometrial and muscular cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive the embryo and support its development to a viable stage. Our findings indicate that BM-MSCs may support uterine tissue regeneration.     

Differential expression of matrix metalloproteinases and interleukin-8 during regeneration of human airway epithelium in vivo

In many airway diseases, the airway epithelium is severely damaged and has to regenerate rapidly to restore its function. The regeneration process involves chronological steps of epithelial cell migration, proliferation, stratification, and differentiation. The present study has used an in vivo humanized airway xenograft model in nude mice that mimics the regeneration dynamics of human airway epithelium after severe injury, and human-specific molecular tools, to study the expression profiles of epithelial matrix metalloproteinases (MMPs)-7 and -9, of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and of the pro-inflammatory cytokine interleukin-8 (IL-8) during the different steps of human airway epithelium regeneration. It was found that during the cell migration and proliferation steps, airway epithelial cells expressed IL-8 at a high level, whereas airway epithelial pseudo-stratification and surface airway epithelial differentiation were associated with increased expression of MMPs and a progressive decrease in IL-8. Interestingly, immunohistochemical analysis revealed exclusive expression of MMPs at the apical part of the well-differentiated regenerated airway epithelium, and incubation of the regenerating epithelial cells with MMP inhibitors led to abnormal epithelial differentiation. These data provide new insight into the temporal expression of MMPs and IL-8 during the regeneration of airway epithelium and demonstrate the involvement of these factors during the different steps that lead to restoration of a well-differentiated and functional airway epithelium.     

Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model

There exists a strong clinical need for a more capable and robust method to achieve bone augmentation, and a system with fine-tuned delivery of demineralized bone matrix (DBM) has the potential to meet that need. As such, the objective of the present study was to investigate a synthetic biodegradable hydrogel for the delivery of DBM for bone augmentation in a rat model. Oligo(poly(ethylene glycol) fumarate) (OPF) constructs were designed and fabricated by varying the content of rat-derived DBM particles (either 1:3, 1:1 or 3:1 DBM:OPF weight ratio on a dry basis) and using two DBM particle size ranges (50–150 or 150–250 μm). The physical properties of the constructs and the bioactivity of the DBM were evaluated. Selected formulations (1:1 and 3:1 with 50–150 μm DBM) were evaluated in vivo compared to an empty control to investigate the effect of DBM dose and construct properties on bone augmentation. Overall, 3:1 constructs with higher DBM content achieved the greatest volume of bone augmentation, exceeding 1:1 constructs and empty implants by 3- and 5-fold, respectively. As such, we have established that a synthetic, biodegradable hydrogel can function as a carrier for DBM, and that the volume of bone augmentation achieved by the constructs correlates directly to the DBM dose.     

骨髓间充质干细胞-脱细胞真皮基质对大鼠表皮缺损的修复

目的研究骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)-脱细胞真皮基质(acellular dermal matrix,ADM)对SD大鼠皮肤表皮缺损的修复。方法培养SD大鼠骨髓细胞,增殖提纯传代至4代时,成骨成脂,流式细胞仪检测鉴定BMSC,然后将其接种到ADM,2d后移植到皮肤表皮缺损的SD大鼠体内,连续5周观察其修复效果。结果 BMSC-ADM修复SD大鼠皮肤表皮缺损效果良好,5周后,肉眼可见材料与组织结合紧密,有弹性,颜色接近皮肤。结论 BMSC-ADM可以作为修复SD大鼠皮肤表皮缺损的组织工程材料。     

可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损

背景：自体髂骨移植一直被认为是骨缺损修复的“金标准”,但其来源有限。 目的：验证应用可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损的效果。 方法：80只雄性SD大鼠建立双侧颅骨缺损模型,随机分为3组：骨修复材料+骨碎补总黄酮组采用可注射骨修复材料结合骨碎补总黄酮灌胃修复大鼠颅骨缺损;骨修复材料+去离子水组采用可注射骨修复材料结合去离子水灌胃修复大鼠颅骨缺损;羟基磷灰石+去离子水组采用羟基磷灰石结合去离子水灌胃修复大鼠颅骨缺损,1次/d,持续8周。于建模后2,4,8周取颅骨标本进行苏木精-伊红染色和Masson染色组织学观察。 结果与结论：羟基磷灰石组新骨形成和材料降解速度较慢;可注射骨修复材料组新骨形成和材料降解较羟基磷灰石组快,利于血管及纤维组织长入;骨碎补总黄酮灌胃可以促进血管及纤维组织长入材料,促进成骨。与羟基磷灰石相比,可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损,可促进新骨形成,缩短骨缺损修复时间。     

Secretion of osteopontin by macrophages and its accumulation at tissue surfaces during wound healing in mineralized tissues: A potential requirement for macrophage adhesion and phagocytosis

Osteopontin (OPN), a noncollagenous, extracellular matrix sialoprotein found at relatively high levels in both normal and pathological mineralized tissues, is expressed by tissue-specific cells in bone, calcified cartilage, and teeth. On the other hand, a hallmark of OPN expression in pathologically mineralizing tissues, and in other soft tissues experiencing a more generalized type of necrotic injury, is the production of OPN by macrophages at the lesion site. In the present study, we have localized OPN and other noncollagenous proteins by ultrastructural colloidal-gold immunocytochemistry using a rat model in which mineralized tissue defects are surgically created in mandibular bone and teeth. The healing response was examined by immunocytochemistry and transmission electron microscopy at 10 min, 3 days and 7 days post-surgery using antibodies against OPN, bone sialoprotein, osteocalcin, bone acidic glycoprotein-75, fibronectin, and amelogenin. Whereas most of these proteins were characteristically distributed within their respective extracellular matrices as described previously, OPN was additionally observed to accumulate as a lamina limitans at surgically exposed bone and tooth surfaces, as well as at the surface of particulate, mineralized tissue debris. Intracellular labeling of the Golgi apparatus and secretory granules of macrophages at the lesion site demonstrated that OPN production by macrophages was a prominent secretory event of the inflammatory response during wound healing in mineralized tissues. Pseudopodal and lamellipodal cytoplasmic extensions of macrophages were observed in direct contact with the OPN-containing lamina limitans at these surfaces. Particulate, calcified debris internalized by macrophages also displayed a prominent surface “coating” of OPN. In conclusion, our interpretation of the present data is that OPN secreted by macrophages may serve as a macrophage adhesion protein, and where concentrated at the surface of small particulate, mineralized tissue debris, may act as an opsonin, thereby facilitating cell adhesion and phagocytosis by macrophages, a process likely mediated by integrin-binding, signal transduction, and cytoskeletal restructuring. © 1996 Wiley-Liss, Inc.     

Effects of short-term treatment with the bisphosphonates zoledronate and pamidronate on rat bone: A comparative histomorphometric study on the cancellous bone formed before,during, and after treatment

To study the anti-resorptive effects of zoledronate and pamidronate on growing long bones we have performed a histomorphometric analysis of the three regions of the proximal tibial cancellous bone of bone formed before, during, and after drug treatment. Male rats (190–220 g) were treated subcutaneously for 10 days with zoledronate (0.028–2.8 μg/kg) or pamidronate (3.7–370 μg/kg) and sacrificed 5 days later. To delineate the three regions of cancellous bone, and for dynamic bone histomorphometry, calcein and demeclocycline were injected at various times. Both bisphosphonates caused a dose-dependent suppression of cancellous bone turnover and resorption to produce an increase in cancellous bone, but zoledronate was 100 times more potent than pamidronate. The increase in the bone amount and connectivity was more pronounced in the bone formed during treatment where transient bone resorption and normal bone formation led to a positive bone balance. In the bone formed before treatment, inhibition of bone resorption associated with reduced bone formation produced a net gain in amount of bone. Although both bone regions showed a positive bone balance, more bone accumulated in the bone formed during treatment probably because its trabecular bone surface was three times greater. In the primary spongiosa formed after treatment, a moderate increase in the bone amount and connectivity was observed only at the highest dose of both bisphosphonates. The bone formed before, during, and after treatment with bisphosphonates responds differently due to differences in bone architecture, rates of modeling and remodeling, and period of drug exposure. Anat. Rec. 249:458–468, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of extracellular matrix proteins during wound healing in the lamina propria of vocal fold in a canine model: A long-term and consecutive study

The characterization of vocal fold wound healing can be reflected by the changes of extracellular matrix (ECM) proteins in the lamina propria. Although the expression of ECM proteins after vocal fold injury has been widely studied, such observations have lacked time continuity and integrity of marker proteins. In this study, we observed the morphology of injured vocal folds in a canine model. We used immunofluorescence staining to evaluate the expression and distribution of ECM proteins, such as collagen, elastin, hyaluronic acid, decorin and fibronectin, from 15 days to 6 months after injury. The results showed that large amounts of ECM proteins were secreted 15–40 days after injury. Collagen and fibronectin secretion increased significantly, and were disorderly deposited. The secretion of decorin and elastin increased slightly, while hyaluronic acid decreased. The 15–40 day post-injury period may be the critical intervention stage in wound healing of vocal folds. From 3 to 6 months after injury, the secretion of ECM proteins declined. However, collagen and fibronectin secretion were still significantly higher than normal with irregular arrangement, while the secretion of elastin, hyaluronic acid and decorin decreased significantly at 6 months. This led to vocal fold inelasticity and stiffness, which required effective long-term interventions to treat scar formation.     

Immunolocalization of vacuolar‐type H+‐ATPase,cathepsin K,matrix metalloproteinase‐9, and receptor activator of NFkB ligand in odontoclasts during physiological root resorption of human deciduous teeth

To investigate the cellular mechanisms of physiological root resorption in human deciduous teeth, the authors examined the immunocytochemical localization of vacuolar‐type H⁺‐ATPase, a lysosomal cysteine proteinase, cathepsin K, matrix metalloproteinase‐9 (MMP‐9), and receptor activator of NFKB ligand (RANKL) in odontoclasts. H⁺‐ATPase, cathepsin K, and MMP‐9 are the most important enzymes for decalcification of apatite crystals and degradation of type‐I collagen. In addition, RANKL is one of the key regulatory molecules in osteoclast formation and functions. Odontoclasts developed extensive ruffled borders and clear zones apposed to the resorbing root dentine surfaces. On immunoelectron microscopy, the expression of vacuolar‐type H⁺‐ATPase was detected along the limiting membranes of pale vacuoles and the ruffled border membranes of odontoclasts. Cathepsin K in odontoclasts was localized within pale vacuoles, lysosomes, the extracellular canals of ruffled borders, and the underlying resorbing dentine surfaces. MMP‐9 localization in odontoclasts was similar to those of cathepsin K. RANKL was detected in both mononuclear stromal cells and odontoclasts located on resorbing dentine surfaces. These results suggest that (1) odontoclasts are directly involved in decalcification of apatite crystals by active extrusion of proton ions mediated by H⁺‐ATPase and (2) extracellular degradation of dentine type‐I collagen by both cathepsin K and MMP‐9, and (3) odontoclast differentiation and activity are regulated, at least in part, by RANKL, possibly produced by mononuclear stromal cells and odontoclasts themselves in the resorbing tissues. Thus, the cellular mechanisms of physiological root resorption appear to be quite similar to those of osteoclastic bone resorption. Anat Rec 264:305–311, 2001. © 2001 Wiley‐Liss, Inc.     

Localization of tartrate-resistant acid phosphatase (TRAP), membrane type-1 matrix metalloproteinases (MT1-MMP) and macrophages during early endochondral bone formation

Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate-resistant acid phosphatase (TRAP) and membrane type-1 matrix metalloproteinases (MT1-MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP-positive chondroclasts resorbed the cartilage matrix. However, occasionally co-expression of TRAP and MT1-MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1-MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1-MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone-lining cells. Considering the well-known fact that not all osteoblasts transform into osteocytes, and in accordance with the present data, we suggest that MT1-MMP is needed at the very beginning of osteocytogenesis and may additionally determine whether an osteoblast further differentiates into an osteocyte.     

Synergy between IL-6 and soluble IL-6 receptor enhances bone morphogenetic protein-2/absorbable collagen sponge-induced bone regeneration via regulation of BMPRIA distribution and degradation

Bone morphogenetic protein-2/absorbable collagen sponge (BMP-2/ACS) implants have been approved for clinical use to induce bone regeneration. We previously showed that exaggerated inflammation characterized by elevated level of inflammatory cytokines including TNF-α, IL-1β, and IL-6 has been shown to inhibit BMP-2/ACS-induced bone regeneration. Furthermore, unlike the negative effects of TNF-α and IL-1β, IL-6 seemed not to affect BMP-2-induced osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs). We hypothesized that there may be a regulatory loop between IL-6 and BMP-2 singling to affect BMP-2/ACS-induced bone regeneration. Here, we established a BMP-2/ACS-induced ectopic bone formation model in rats and fund that IL-6 injection significantly increased BMP-2/ACS-induced bone mass. Consistent with this animal model, an in vitro study demonstrated that synergy between IL-6 and soluble IL-6 receptor (IL-6/sIL-6R) promotes BMP-2-induced osteoblastic differentiation of human BMSCs through amplification of BMP/Smad signaling. Strikingly, IL-6 injection did not activate osteoclast-mediated bone resorption in the ectopic bone formation model, and IL-6/sIL-6R treatment did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastic differentiation of human peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, IL-6/sIL-6R treatment did not affect expression of BMP receptors, but enhanced the cell surface translocation of BMP receptor IA (BMPRIA) and inhibited the degradation of BMPRIA. Collectively, these findings indicate that synergy between IL-6 and sIL-6R promotes the cell surface translocation of BMPRIA and maintains the stability of BMPRIA expression, leading to enhanced BMP-2/ACS-induced bone regeneration.