Appropriate pore size for bone formation potential of porous collagen type I-based recombinant peptide.

Introduction: In this study, we developed porous medium cross-linked recombinant collagen peptide (mRCP) with two different ranges of interconnected pore sizes, Small-mRCP (S-mRCP) with a range of 100-300 µm and Large-mRCP (L-mRCP) with a range of 200-500 µm, to compare the effect of pore size on bone regeneration in a calvarial bone defect. Methods: Calvarial bone defects were created in Sprague-Dawley rats through a surgical procedure. The rats were divided into 2 groups: S-mRCP implanted group and L-mRCP implanted group. The newly formed bone volume and bone mineral density (BMD) was evaluated by micro-computed tomography (micro-CT) immediately after implantation and at 1, 2, 3, and 4 weeks after implantation. In addition, histological analyses were carried out with hematoxylin and eosin (H&E) staining at 4 weeks after implantation to measure the newly formed bone area between each group in the entire defect, as well as the central side, the two peripheral sides (right and left), the periosteal (top) side and the dura matter (bottom) side of the defect. Results: Micro-CT analysis showed no significant differences in the amount of bone volume between the S-mRCP and L-mRCP implanted groups at 1, 2, 3 and 4 weeks after implantation. BMD was equivalent to that of the adjacent native calvaria bone at 4 weeks after implantation. H&E images showed that the newly formed bone area in the entire defect was significantly larger in the S-mRCP implanted group than in the L-mRCP implanted group. Furthermore, the amount of newly formed bone area in all sides of the defect was significantly more in the S-mRCP implanted group than in the L-mRCP implanted group. Conclusion: These results indicate that the smaller pore size range of 100-300 µm is appropriate for mRCP in bone regeneration.

Bone formation potential of collagen type I-based recombinant peptide particles in rat calvaria defects.

INTRODUCTION: This study aimed to examine the bone-forming ability of medium-cross-linked recombinant collagen peptide (mRCP) particles developedbased on human collagen type I, contains an arginyl-glycyl-aspartic acid-rich motif, fabricated as bone filling material, compared to that of the autologous bone graft. METHODS: Calvarial bone defects were created in immunodeficient rats though a surgical procedure. The rats were divided into 2 groups: mRCP graft and tibia bone graft (bone graft). The bone formation potential of mRCP was evaluated by micro-computed tomography and hematoxylin-eosin staining at 1, 2, 3, and 4 weeks after surgery, and the data were analyzed and compared to those of the bone graft. RESULTS: The axial volume-rendered images demonstrated considerable bony bridging with the mRCP graft, but there was no significant difference in the bone volume and bone mineral density between the mRCP graft and bone graft at 4 weeks. The peripheral new bone density was significantly higher than the central new bone density and the bottom side score was significantly higher than the top side score at early stage in the regenerated bone within the bone defects. CONCLUSION: These results indicate that mRCP has a high potential of recruiting osteogenic cells, comparable to that of autologous bone chips.

Optimization of culture duration of bone marrow cells before transplantation with a ß-tricalcium phosphate/recombinant collagen peptide hybrid scaffold.

INTRODUCTION: Currently, various kinds of materials are used for the treatment of bone defects. In general, these materials have a problem of formativeness. The three -dimensional (3D) printing technique has been introduced to fabricate artificial bone with arbitrary shapes, but poor bone replacement is still problematic.Our group has created a ß⁻tricalcium phosphate (ß⁻TCP) scaffold by applying 3D printing technology. This scaffold has an arbitrary shape and an internal structure suitable for cell loading, growth, and colonization. The scaffold was coated with a recombinant collagen peptide (RCP) to promote bone replacement.As indicated by several studies, cells loaded to scaffolds promote bone regeneration, especially when they are induced osteoblastic differentiation before transplantation. In this study, culture duration for bone marrow cells was optimized before being loaded to this new scaffold material. METHOD: Bone marrow cells isolated from C57BL/6J mice were subjected to osteogenic culture for 4, 7, and 14 days. The differentiation status of the cells was examined by alkaline phosphatase staining, alizarin red staining, and real-time RT-PCR for differentiation markers. In addition, the flow of changes in the abundance of endothelial cells and monocytes was analyzed by flow cytometry according to the culture period of bone marrow cells.Next, cells at days 4, 7, and 14 of culture were placed on a ß-TCP/RCP scaffold and implanted subcutaneously into the back of C57BL/6J mice. Grafts were harvested and evaluated histologically 8 weeks later. Finally, Cells cultured for 7 days were also transplanted subperiosteally in the skull of the mouse with scaffolds. RESULT: Alkaline phosphatase staining was most prominent at 7 days, and alizarin red staining was positive at 14 days. Real-time RT-PCR revealed that Runx2 and Alp peaked at 7 days, while expression of Col1a1 and Bglap was highest at 14 days. Flow cytometry indicated that endothelial cells increased from day 0 to day 7, while monocytes increased continuously from day 0 to day 14. When transplanted into mice, the scaffold with cells cultured for 7 days exhibited the most prominent osteogenesis. The scaffold, which was transplanted subperiosteally in the skull, retained its shape and was replaced with regenerated bone over a large area of the field of view. CONCLUSION: Osteoblasts before full maturation are most efficient for bone regeneration, and the pre-culture period suitable for cells to be loaded onto a ß-TCP/RCP hybrid scaffold is approximately 7 days.This ß-TCP/RCP hybrid scaffolds will also be useful for bone augmentation.

* Biomineralized Recombinant Collagen-Based Scaffold Mimicking Native Bone Enhances Mesenchymal Stem Cell Interaction and Differentiation.

The need of synthetic bone grafts that recreate from macro- to nanoscale level the biochemical and biophysical cues of bone extracellular matrix has been a major driving force for the development of new generation of biomaterials. In this study, synthetic bone substitutes have been synthesized via biomimetic mineralization of a recombinant collagen type I-derived peptide (RCP), enriched in tri-amino acid sequence arginine-glycine-aspartate (RGD). Three-dimensional (3D) isotropic porous scaffolds of three different compositions are developed by freeze-drying: non-mineralized (RCP, as a control), mineralized (Ap/RCP), and mineralized scaffolds in the presence of magnesium (MgAp/RCP) that closely imitate bone composition. The effect of mineral phase on scaffold pore size, porosity, and permeability, as well as on their in vitro kinetic degradation, is evaluated. The ultimate goal is to investigate how chemical (i.e., surface chemistry and ion release from scaffold) together with physical signals (i.e., surface nanotopography) conferred via biomimetic mineralization can persuade and guide mesenchymal stem cell (MSC) interaction and fate. The three scaffold compositions showed optimum pore size and porosity for osteoconduction, without significant differences between them. The degradation tests confirmed that MgAp/RCP scaffolds presented higher reactivity under physiological condition compared to Ap/RCP ones. The in vitro study revealed an enhanced cell growth and proliferation on MgAp/RCP scaffolds at day 7, 14, and 21. Furthermore, MgAp/RCP scaffolds potentially promoted cell migration through the inner areas reaching the bottom of the scaffold after 14 days. MSCs cultured on MgAp/RCP scaffolds displayed higher gene and protein expressions of osteogenic markers when comparing them with the results of those MSCs grown on RCP or Ap/RCP scaffolds. This work highlights that mineralization of recombinant collagen mimicking bone mineral composition and morphology is a versatile approach to design smart scaffold interface in a 3D model guiding MSC fate.