Non-critical Size Defects Research Articles

Polychrome labeling of bone with seven different fluorochromes: Enhancing fluorochrome discrimination by spectral image analysis

Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel). Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrome suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 μm thick sections could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrome without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions.

Does platelet‐rich plasma promote remodeling of autologous bone grafts used for augmentation of the maxillary sinus floor?

The aim of this study was to evaluate the effect of platelet-rich plasma (PRP) on remodeling of autologous bone grafts used for augmentation of the floor of the maxillary sinus. In five edentulous patients suffering from insufficient retention of their upper denture related to a severely resorbed maxilla, the floor of both maxillary sinus was augmented with an autologous bone graft from the iliac crest. Randomly, PRP was added to the bone graft used to augment the floor of the left or right sinus (split-mouth design). Three months after the reconstruction, bone biopsies were taken with a trephine from the planned implant sites (N=30). Subsequently, three implants were placed in the left and right posterior maxilla. Microradiograms were made of all biopsies (N=30), whereafter the biopsies were processed for light microscopic examination. In addition, clinical parameters were scored. Wound healing was uneventful, clinically no difference was observed between the side treated with PRP or not. Also microradiographical and histomorphological examination of the biopsies revealed no statistical difference between the PRP- and non-PRP side. One implant placed in the PRP side of the graft was lost during the healing phase. Implant-retained overdentures were fabricated 6 months after implantation. All patients functioned well (follow-up 20.2+/-4.3 months). In this study, no beneficial effect of PRP on wound healing and bone remodeling was observed. It is posed that PRP has no additional value in promoting healing of grafted non-critical size defects.

Molecular Biological Evaluation of Bioactive Glass Microspheres and Adjunct Bone Morphogenetic Protein 2 Gene Transfer in the Enhancement of New Bone Formation

Bioactive glass is a promising osteoconductive silica-based biomaterial for guidance of new bone growth. On the basis of several in vitro studies, the material appears able to promote osteoblast functions. In our in vivo study, the osteopromotive effect of bioactive glass microspheres seemed to surpass the osteoinductive action of direct adenovirus-mediated human bone morphogenetic protein 2 (BMP-2) gene transfer in a noncritical size bone defect model. The current study was initiated to elucidate the molecular mechanism behind bioactive glass action with or without adjunct BMP-2 gene transfer. A standardized bone defect of the rat tibia was filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (RAdBMP-2). Control defects were left empty or filled with bioactive glass microspheres with injection of adenovirus carrying the lacZ reporter gene or saline. Quantitative polymerase chain reaction confirmed the expression of the transferred human BMP-2 gene at the defect area at 4 days, but not in intact reference tissues. Bone matrix components (collagens I, II, and III, osteocalcin, osteonectin, and osteopontin) and resorption markers (cathepsin K and MMP-9), determined by Northern analysis, showed a completely different pattern of gene expression in defects filled with bioactive glass compared with control defects left to heal without filling. Bioactive glass induced a long-lasting production of bone matrix with concurrent upregulation of osteoclastic markers, a sign of high bone turnover. Combining RAdBMP-2 gene transfer with bioactive glass decelerated the high turnover, but did not influence the balance of synthesis and resorption. This molecular analysis confirmed not only the highly osteopromotive effect of bioactive glass microspheres, but also the accelerated rate of new bone resorption on its surface. At least in noncritical size defects this impact of bioactive glass seems to saturate new bone formation on its surface and thereby overshadow the effect of BMP-2 gene transfer.

Bone defect repair in immobilization-induced osteopenia: a pQCT, biomechanical, and molecular biologic study in the mouse femur

The present study was carried out to determine whether immobilization-induced (Im) osteopenic bone possesses the same reparative capacity as normal healthy bone. Furthermore, the effects of mechanical loading versus immobilization on bone defect healing were studied. Three-week cast-immobilization was used to induce local osteopenia in mice. A standardized metaphyseal bone defect of the distal femur was created unilaterally both in immobilization-induced (Im) osteopenic mice and in nonimmobilized (Mo) age-matched control animals. After creation of the bone defect, the animals in both groups were further divided into two groups: 3-week cast-immobilization (Im-Im and Mo-Im) groups, and unrestricted weight-bearing (Im-Mo and Mo-Mo) groups. The healing process was followed up to 3 weeks using RNA analysis, histomorphometry, biomechanical testing, and pQCT measurements. At 3 weeks of healing without immobilization, bone mineral density (BMD), as well as bone bending stiffness and strength were higher in normal (Mo-Mo) than in osteopenic (Im-Mo) bone. Although the levels of mRNAs characteristic to chondrocytes (Sox9 and type II collagen), hypertrophic chondrocytes (Type X collagen), osteoblasts (type I collagen and osteocalcin), and osteoclasts (cathepsin K) during the bone defect healing exhibited similarities in their expression profiles, mechanical loading conditions also caused characteristic differences. Mechanical loading during healing (Mo-Mo group) induced stronger expression of cartilage- and bone-specific genes and resulted in higher BMD than that seen in the cast-immobilized group (Mo-Im). In biomechanical analysis, increased bending stiffness and strength were also observed in animals that were allowed weight-bearing during healing. Thus, our study shows that bone healing follows the same molecular pathway both in osteopenic and normal bones and presents evidence for reduced or delayed regeneration of noncritical size defects in immobilization-induced osteopenic bone.

TCP is hardly resorbed and not osteoconductive in a non-loading calvarial model

Tricalciumphosphate (TCP) has been used as a ceramic bone substitute material in the orthopedic field as well as in craniofacial surgery. Some controversies exist concerning the osteoconductive potential of this material in different implantation sites. This study was designed to evaluate the biological response of calvarial bone towards TCP granules under non-loading conditions to assess the potential of TCP as a biodegredable and osteoconductive bone substitue material for the cranial vault. Full-thickness non-critical size defects were made bilaterally in the calvaria of 21 adult Wistar rats. One side was filled by TCP granules, the contralateral side was left empty and used as a control. Animals were sacrified in defined time intervals up to 6 months. Bone regeneration was analyzed with special respect toward the micromorphological and microanalytical features of the material–bone interaction by electron microscopy and electron diffraction analysis. Histologic examination revealed no TCP degradation even after 6 months of implantation. In contrast, a nearly complete bone regeneration of control defects was found after 6 months. At all times TCP was surrounded by a thin fibrous layer without presence of osteoblasts and features of regular mineralization. As far as degradation and substitution are concerned, TCP is a less favourable material under conditions of non-loading.