Osteogenic differentiation of adipose-derived canine mesenchymal stem cells seeded in porous calcium-phosphate scaffolds.

Introduction: Engineered bone graft substitutes are a promising alternative and supplement to autologous bone grafts as treatments for bone healing impairment. Advances in human medicine extend an invitation to pursue these biomimetic strategies in animal patients, substantiated by the theory that specialized scaffolds, multipotent cells, and biological cues may be combined into a bioactive implant intended for the enhancement of tissue regeneration. Methods: This proof-of-concept study was designed to evaluate and validate the feasibility of beta-tricalcium phosphate foam scaffolds seeded with canine mesenchymal stem cells derived from adipose tissue. Cell-inoculated samples and sham controls were cultured statically for 72 hours in complete growth medium to evaluate seeding capacity, while a subset of loaded scaffolds was further induced with osteogenic culture medium for 21 days. Produced implants were characterized and validated with a combination of immunofluorescence and reflection confocal microscopy, scanning electron microscopy, and polymerase chain reaction to confirm osteogenic differentiation in tridimensional-induced samples. Results: After 72 hours of culture, all inoculated scaffolds presented widespread yet heterogeneous surface seeding, distinctively congregating stem cells around pore openings. Furthermore, at 21 days of osteogenic culture conditions, robust osteoblastic differentiation of the seeded cells was confirmed by the change of cell morphology and evident deposition of extra-cellular matrix, accompanied by mineralization and scaffold remodeling; furthermore, all induced cell-loaded implants lost specific stemness immunophenotype expression and simultaneously upregulated genomic expression of osteogenic genes Osterix and Ostecalcin. Conclusions: ß-TCP bio-ceramic foam scaffolds proved to be suitable carriers and hosts of canine adipose-derived MSCs, promoting not only surface attachment and proliferation, but also demonstrating strong in-vitro osteogenic potential. Although this research provides satisfactory in-vitro validation for the conceptualization and feasibility of a canine bio-active bone implant, further testing such as patient safety, large-scale reproducibility, and quality assessment are needed for regulatory compliance in future commercial clinical applications.

On-Growth and In-Growth Osseointegration Enhancement in PM Porous Ti-Scaffolds by Two Different Bioactivation Strategies: Alkali Thermochemical Treatment and RGD Peptide Coating.

A lack of primary stability and osteointegration in metallic implants may result in implant loosening and failure. Adding porosity to metallic implants reduces the stress shielding effect and improves implant performance, allowing the surrounding bone tissue to grow into the scaffold. However, a bioactive surface is needed to stimulate implant osteointegration and improve mechanical stability. In this study, porous titanium implants were produced via powder sintering to create different porous diameters and open interconnectivity. Two strategies were used to generate a bioactive surface on the metallic foams: (1) an inorganic alkali thermochemical treatment, (2) grafting a cell adhesive tripeptide (RGD). RGD peptides exhibit an affinity for integrins expressed by osteoblasts, and have been reported to improve osteoblast adhesion, whereas the thermochemical treatment is known to improve titanium implant osseointegration upon implantation. Bioactivated scaffolds and control samples were implanted into the tibiae of rabbits to analyze the effect of these two strategies in vivo regarding bone tissue regeneration through interconnected porosity. Histomorphometric evaluation was performed at 4 and 12 weeks after implantation. Bone-to-implant contact (BIC) and bone in-growth and on-growth were evaluated in different regions of interest (ROIs) inside and outside the implant. The results of this study show that after a long-term postoperative period, the RGD-coated samples presented higher quantification values of quantified newly formed bone tissue in the implant's outer area. However, the total analyzed bone in-growth was observed to be slightly greater in the scaffolds treated with alkali thermochemical treatment. These results suggest that both strategies contribute to enhancing porous metallic implant stability and osteointegration, and a combination of both strategies might be worth pursuing.

Use of three-dimensionally printed ß-tricalcium phosphate synthetic bone graft combined with recombinant human bone morphogenic protein-2 to treat a severe radial atrophic nonunion in a Yorkshire terrier.

OBJECTIVE: To describe a novel surgical approach to treat a critical-sized bone defect due to severe, radial atrophic nonunion in a miniature dog. STUDY DESIGN: Case report ANIMAL: A 1-year-old Yorkshire terrier with a critical-sized left radial defect after failed internal fixation of a transverse radial fracture. METHODS: Computed tomographic (CT) images of the radius were imported for three-dimensional (3D) printing of a custom-designed synthetic 3D-printed ß-tricalcium phosphate (ß-TCP) scaffold. The radius was exposed, and the ß-TCP scaffold was press-fitted in the bone gap underneath the plate. Recombinant human bone morphogenic protein-2 (RhBMP-2) collagen sponges were squeezed to soak the scaffold with growth factor and then placed on both sides of the synthetic graft. Two additional cortical screws were also placed prior to routine closure of the surgical site. RESULTS: Radiographic examination was consistent with complete healing of the radius defect 4 months after surgery. The bone plate was removed 10 months after surgery. According to CT examination 18 months after surgery, there was no evidence of the synthetic graft; instead, complete corticalization of the affected area was noted. Complete functional recovery was observed until the last clinical follow-up 36 months postoperatively. CONCLUSION: Screw fixation and use of a 3D-printed ceramic scaffold augmented with rhBMP-2 resulted in excellent bone regeneration of the nonunion and full recovery of a miniature breed dog. CLINICAL SIGNIFICANCE: The therapeutic approach used in this dog could be considered as an option for treatment of large-bone defects in veterinary orthopedics, especially for defects affecting the distal radius of miniature dogs.

Two Different Strategies to Enhance Osseointegration in Porous Titanium: Inorganic Thermo-Chemical Treatment Versus Organic Coating by Peptide Adsorption.

In this study, highly-interconnected porous titanium implants were produced by powder sintering with different porous diameters and open interconnectivity. The actual foams were produced using high cost technologies: Chemical Vapor Deposition (CVD), Physical Vapor Deposition (PVD), and spark plasma sintering, and the porosity and/or interconnection was not optimized. The aim was to generate a bioactive surface on foams using two different strategies, based on inorganic thermo-chemical treatment and organic coating by peptide adsorption, to enhance osseointegration. Porosity was produced using NaCl as a space holder and polyethyleneglicol as a binder phase. Static and fatigue tests were performed in order to determine mechanical behaviors. Surface bioactivation was performed using a thermo-chemical treatment or by chemical adsorption with peptides. Osteoblast-like cells were cultured and cytotoxicity was measured. Bioactivated scaffolds and a control were implanted in the tibiae of rabbits. Histomorphometric evaluation was performed at 4 weeks after implantation. Interconnected porosity was 53% with an average diameter of 210 µm and an elastic modulus of around 1 GPa with good mechanical properties. The samples presented cell survival values close to 100% of viability. Newly formed bone was observed inside macropores, through interconnected porosity, and on the implant surface. Successful bone colonization of inner structure (40%) suggested good osteoconductive capability of the implant. Bioactivated foams showed better results than non-treated ones, suggesting both bioactivation strategies induce osteointegration capability.

Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture.

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.

Osteoinduction by Foamed and 3D-Printed Calcium Phosphate Scaffolds: Effect of Nanostructure and Pore Architecture.

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.