Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats

OBJECTIVES To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.

Evaluation of a new experimental model to study bone healing after ridge expansion with simultaneous implant placement--a pilot study in minipigs.

OBJECTIVE To evaluate the suitability of a minipig model for the study of bone healing and osseointegration of dental implants following bone splitting and expansion of narrow ridges. MATERIAL AND METHODS In four minipigs, the mandibular premolars and first molars were extracted together with removal of the buccal bone plate. Three months later, ridge splitting and expansion was performed with simultaneous placement of three titanium implants per quadrant. On one side of the mandible, the expanded bone gap between the implants was filled with an alloplastic biphasic calcium phosphate (BCP) material, while the gap on the other side was left unfilled. A barrier membrane was placed in half of the quadrants. After a healing period of 6 weeks, the animals were sacrificed for histological evaluation. RESULTS In all groups, no bone fractures occurred, no implants were lost, all 24 implants were osseointegrated, and the gap created by bone splitting was filled with new bone, irrespective of whether BCP or a barrier membrane was used. Slight exposure of five implants was observed, but did not lead to implant loss. The level of the most coronal bone-to-implant contact varied without being dependent on the use of BCP or a barrier membrane. In all groups, the BCP particles were not present deep in the bone-filled gap. However, BCP particles were seen at the crestal bone margin, where they were partly integrated in the new bone. CONCLUSIONS This new minipig model holds great promise for studying experimental ridge splitting/expansion. However, efforts must be undertaken to reduce implant exposure and buccal bone resorption.

Bone healing around nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, and autogenous bone in mandibular bone defects.

The individual healing profile of a given bone substitute with respect to osteogenic potential and substitution rate must be considered when selecting adjunctive grafting materials for bone regeneration procedures. In this study, standardized mandibular defects in minipigs were filled with nanocrystalline hydroxyapatite (HA-SiO), deproteinized bovine bone mineral (DBBM), biphasic calcium phosphate (BCP) with a 60/40% HA/β-TCP (BCP 60/40) ratio, or particulate autogenous bone (A) for histological and histomorphometric analysis. At 2 weeks, percent filler amongst the test groups (DBBM (35.65%), HA-SiO (34.47%), followed by BCP 60/40 (23.64%)) was significantly higher than the more rapidly substituted autogenous bone (17.1%). Autogenous bone yielded significantly more new bone (21.81%) over all test groups (4.91%-7.74%) and significantly more osteoid (5.53%) than BCP 60/40 (3%) and DBBM (2.25%). At 8 weeks, percent filler amongst the test groups (DBBM (31.6%), HA-SiO (31.23%), followed by BCP 60/40 (23.65%)) demonstrated a similar pattern and was again significantly higher as compared to autogenous bone (9.29%). Autogenous bone again exhibited statistically significantly greater new bone (55.13%) over HA-SiO (40.62%), BCP 60/40 (40.21%), and DBBM (36.35%). These results suggest that the osteogenic potential of HA-SiO and BCP is inferior when compared to autogenous bone. However, in instances where a low substitution rate is desired to maintain the volume stability of augmented sites, particularly in the esthetic zone, HA-SiO and DBBM may be favored. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1478-1487, 2015.

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing. © 2014 Springer-Verlag.

Improved healing of large segmental defects in the rat femur by reverse dynamization in the presence of bone morphogenetic protein-2

Background Large segmental defects in bone do not heal well and present clinical challenges. This study investigated modulation of the mechanical environment as a means of improving bone healing in the presence of bone morphogenetic protein (BMP)-2. Although the influence of mechanical forces on the healing of fractures is well established, no previous studies, to our knowledge, have described their influence on the healing of large segmental defects. We hypothesized that bone-healing would be improved by initial, low-stiffness fixation of the defect, followed by high-stiffness fixation during the healing process. We call this reverse dynamization. Methods A rat model of a critical-sized femoral defect was used. External fixators were constructed to provide different degrees of stiffness and, importantly, the ability to change stiffness during the healing process in vivo. Healing of the critical-sized defects was initiated by the implantation of 11 mg of recombinant human BMP (rhBMP)-2 on a collagen sponge. Groups of rats receiving BMP-2 were allowed to heal with low, medium, and high-stiffness fixators, as well as under conditions of reverse dynamization, in which the stiffness was changed from low to high at two weeks. Healing was assessed at eight weeks with use of radiographs, histological analysis, microcomputed tomography, dual x-ray absorptiometry, and mechanical testing. Results Under constant stiffness, the low-stiffness fixator produced the best healing after eight weeks. However, reverse dynamization provided considerable improvement, resulting in a marked acceleration of the healing process by all of the criteria of this study. The histological data suggest that this was the result of intramembranous, rather than endochondral, ossification. Conclusions Reverse dynamization accelerated healing in the presence of BMP-2 in the rat femur and is worthy of further investigation as a means of improving the healing of large segmental bone defects. Clinical Relevance These data provide the basis of a novel, simple, and inexpensive way to improve the healing of critical-sized defects in long bones. Reverse dynamization may also be applicable to other circumstances in which bonehealing is problematic.

Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics

This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

Healing outcomes at implants installed in sites augmented with particulate autologous bone and xenografts. An experimental study in dogs

Aim: To evaluate the integration of implants installed at the interface of pristine and grafted tissue augmented with particulate autologous bone or deproteinized bovine bone mineral (DBBM), concomitantly with a collagen membrane. Material and methods: In 6 Labrador dogs, the distal root of 3P3 and 4P4 was endodontically treated and hemi-sected, and the mesial roots extracted concomitantly with the extraction of 2P2. The buccal bony walls were removed, and two box-shaped defects, one larger and one smaller, were created. After 3 months, flaps were elevated, and the defects were filled with particulate autologous bone or DBBM in the right and left side of the mandible, respectively. Collagen membranes were used to cover the grafted areas. Three months later, flaps were elevated, and a customized device was used as surgical guide to prepare the recipient sites at the interface between grafts and pristine bone. One implant was installed in each of the four defects. After 3 months, biopsies were harvested and ground sections prepared for histological evaluation. Results: The augmentation technique was effective at all sites and all the foreseen implants were installed. In the histological analysis, all implants were integrated in mature bone, at both the buccal and lingual aspects. The most coronal bone-to-implant contact and the top of the buccal bony crest were located at a similar distance between test and control implants. However, these distances were higher at the larger compared with the smaller defects. Especially in the large defect, residual particles of DBBM were found embedded into connective tissue and located outside the bony crest. Conclusions: Particulate autologous bone as well as DBBM particles used to augment horizontally the alveolar bony process allowed for the osseointegration of implants installed after 3 months of healing. © 2012 John Wiley & Sons A/S.

Healing of normal and osteopenic bone with titanium implant and low-level laser therapy (GaAlAs): a histomorphometric study in rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Healing Process of Autogenous Bone Graft in Spontaneously Hypertensive Rats Treated With Losartan: An Immunohistochemical and Histomorphometric Study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rank protein immunolabeling during bone-implant interface healing process

The purpose of this paper was to evaluate the expression of RANK protein during bone-healing process around machined surface implants. Twenty male Wistar rats, 90 days old, after having had a 2 mm diameter and 6 mm long implant inserted in their right tibias, were evaluated at 7, 14, 21, and 42 days after healing. After obtaining the histological samples, slides were subjected to RANK immunostaining reaction. Results were quantitatively evaluated. Results. Immunolabeling analysis showed expressions of RANK in osteoclast and osteoblast lineage cells. The statistical analysis showed an increase in the expression of RANK in osteoblasts at 7 postoperative days and a gradual decrease during the chronology of the healing process demonstrated by mild cellular activity in the final stage (P < .05). Conclusion. RANK immunolabeling was observed especially in osteoclast and osteoblast cells in primary bone during the initial periods of bone-healing/implant interface.

Periosteal BMP2 activity drives bone graft healing

Bone graft incorporation depends on the orchestrated activation of numerous growth factors and cytokines in both the host and the graft. Prominent in this signaling cascade is BMP2. Although BMP2 is dispensable for bone formation, it is required for the initiation of bone repair; thus understanding the cellular mechanisms underlying bone regeneration driven by BMP2 is essential for improving bone graft therapies. In the present study, we assessed the role of Bmp2 in bone graft incorporation using mice in which Bmp2 has been removed from the limb prior to skeletal formation (Bmp2(cKO)). When autograft transplantations were performed in Bmp2cKO mice, callus formation and bone healing were absent. Transplantation of either a vital wild type (WT) bone graft into a Bmp2(cKO) host or a vital Bmp2(cKO) graft into a WT host also resulted in the inhibition of bone graft incorporation. Histological analyses of these transplants show that in the absence of BMP2, periosteal progenitors remain quiescent and healing is not initiated. When we analyzed the expression of Sox9, a marker of chondrogenesis, on the graft surface, we found it significantly reduced when BMP2 was absent in either the graft itself or the host, suggesting that local BMP2 levels drive periosteal cell condensation and subsequent callus cell differentiation. The lack of integrated healing in the absence of BMP2 was not due to the inability of periosteal cells to respond to BMP2. Healing was achieved when grafts were pre-soaked in rhBMP2 protein, indicating that periosteal progenitors remain responsive in the absence of BMP2. In contrast to the requirement for BMP2 in periosteal progenitor activation in vital bone grafts, we found that bone matrix-derived BMP2 does not significantly enhance bone graft incorporation. Taken together, our data show that BMP2 signaling is not essential for the maintenance of periosteal progenitors, but is required for the activation of these progenitors and their subsequent differentiation along the osteo-chondrogenic pathway. These results indicate that BMP2 will be among the signaling molecules whose presence will determine success or failure of new bone graft strategies.

Simulation of the nutrient supply in fracture healing

The healing process for bone fractures is sensitive to mechanical stability and blood supply at the fracture site. Most currently available mechanobiological algorithms of bone healing are based solely on mechanical stimuli, while the explicit analysis of revascularization and its influences on the healing process have not been thoroughly investigated in the literature. In this paper, revascularization was described by two separate processes: angiogenesis and nutrition supply. The mathematical models for angiogenesis and nutrition supply have been proposed and integrated into an existing fuzzy algorithm of fracture healing. The computational algorithm of fracture healing, consisting of stress analysis, analyses of angiogenesis and nutrient supply, and tissue differentiation, has been tested on and compared with animal experimental results published previously. The simulation results showed that, for a small and medium-sized fracture gap, the nutrient supply is sufficient for bone healing, for a large fracture gap, non-union may be induced either by deficient nutrient supply or inadequate mechanical conditions. The comparisons with experimental results demonstrated that the improved computational algorithm is able to simulate a broad spectrum of fracture healing cases and to predict and explain delayed unions and non-union induced by large gap sizes and different mechanical conditions. The new algorithm will allow the simulation of more realistic clinical fracture healing cases with various fracture gaps and geometries and may be helpful to optimise implants and methods for fracture fixation.

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly 3-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 mg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing microcomputed tomography (mCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non- BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by mCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, microcompression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects. Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.

CYR61 (CCN1) protein expression during fracture healing in an ovine tibial model and its relation to the mechanical fixation stability

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.