We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.

Matrix vesicles (MVs) are well positioned in the growth plate to serve as a carrier of morphogenetic information to nearby chondrocytes and osteoblasts. Bone morphogenetic proteins (BMPs) carried in MVs could promote differentiation of these skeletal cells. Vascular endothelial growth factor (VEGF) in MVs could stimulate angiogenesis. Therefore, a study was undertaken to confirm the presence of bone morphogenetic protein (BMP)-1 through-7, VEGF, and the noncollagenous matrix proteins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) in isolated rat growth plate MVs. MVs were isolated from collagenase-digested rachitic rat tibial and femoral growth plates. The presence of BMP-1 through BMP-7, VEGF, BSP, ON, OPN, and OC was evaluated by Western blot, plus ELISA analyses for BMP-2 and-4 content. The alkaline phosphatase-raising ability of MV extracts on cultured rat growth plate chondrocytes was measured as a reflection of MV ability to promote chondroosseous differentiation. BMP-1 through-7, VEGF, BSP, ON, OPN, and OC were all detected by Western blot analyses. Chondrocytes treated with MV extracts showed a two-to threefold increase in alkaline phosphatase activity over control, indicating increased differentiation. Significant amounts of BMP-2 and BMP-4 were detected in MVs by ELISA. Combined, these data suggest that MVs could play an important morphogenetic role in growth plate and endochondral bone formation.

The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel biomaterials that are able to promote the healing and regeneration of injured bones are needed to overcome the limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-to-batch consistency, among others. Furthermore, they can self-assemble into physically crosslinked hydrogels above a certain transition temperature, in this case body temperature, but are injectable below this temperature, thereby markedly reducing surgical invasiveness. In this study, we have developed two bioactive hydrogel-forming ELRs, one including the osteogenic and osteoinductive bone morphogenetic protein-2 (BMP-2) and the other the Arg-Gly-Asp (RGD) cell adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand white rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neoformation was confirmed using different techniques, such as radiography, computed tomography, and histology. This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.

An ability to induce new bone formation at a required site would represent a considerable advance in bone repair and tissue engineering. It has been shown that the healing of critical-size bone defects in rats can be augmented by extracts of Saos-2 cells. These human osteosarcoma cells uniquely contain a bone-inducing activity, whereas other human osteosarcoma cells, e.g., U-2 OS cells, cannot replicate the osteoinductive capacity. To understand the necessary components of the Saos-2 bone-inducing activity, this study compared osteoinductive Saos-2 cells with non-osteoinductive U-2 OS cells with respect to the synthesis of bone morphogenetic proteins (BMPs)-1, -2, -3, -4, -5, -6, and -7 and the non-collagenous matrix proteins bone sialoprotein (BSP), osteonectin (ON), osteopontin (OPN), and osteocalcin (OC). The main differences were abundant synthesis of BMP-1/tolloid, BMP-3, -4, and BSP by Saos-2 cells, but absence or reduced synthesis in U-2 OS cells. BMP-2 and -7 were present in low amounts in both cell types, while BMP-5 and -6 were more abundant in U-2 OS cells, suggesting that these BMPs were of lesser importance for the osteoinductivity of Saos-2 cells. However, a relatively high expression of BMP-3 and -4, together with BMP-1/tolloid, may be important for the osteoinductive capacity of Saos-2 cells. The inability of U2-OS cells to induce bone, despite expressing most of the BMPs, may be due to an insufficiency of tolloid, BMP-3 or -4, BSP, and/or other unknown factors. A better understanding of the necessary components of the Saos-2 cell bone-inducing agent may, in future, lead to clinically useful Saos-2 cell products for bone repair and tissue engineering.

To induce new bone formation, mesenchymal stem cells were seeded onto atelocollagen honeycomb scaffold. We evaluated the efficacy of this scaffold combined with KUSA/A1 cells in vivo. KUSA/A1 cells alone and with atelocollagen were implanted in the subcutaneous pockets of 4-week-old male SCID mice. The transplants were subjected to radiographical, histological, and immunohistochemical examinations after 2 and 4 weeks of implantation. Radiographically, both KUSA/A1 cells alone and KUSA/A1-atelocollagen showed some radiopaque areas formation but the latter disclosed a larger amount. Histologically, KUSA/A1 cells alone showed few small islands of new bone formation surrounded by a thin layer of cellular proliferation. On the other hand, KUSA/A1-atelocollagen revealed abundant new bone formation as well as cellular proliferation. We also determined the immunolocalization of type I collagen, CD34, osteocalcin, osteopontin, and PCNA in this newly formed bone. Our results indicated that collagen scaffold plays an important role allowing vessel formation and cell anchorage, especially through the proliferation and differentiation process in a confined space. This study supports tissue engineering as an alternative for treating different target diseases, such as trauma or congenital defects, and enhances existing therapeutic applications.