Combination therapy with PTH and DBM cannot heal a critical sized murine femoral defect

Orthopaedic surgeons continue to search for cost‐effective bone graft substitutes to enhance bone repair. Teriparatide (PTH 1‐34) and demineralized bone matrix (DBM) have been used in patients to promote bone healing. We evaluated the efficacy of PTH and DBM in healing a critical sized femoral defect in three lineage‐specific transgenic mice expressing Col3.6GFPtopaz (pre‐osteoblastic marker), Col2.3GFPemerald (osteoblastic marker) and α‐SMA‐Cherry (pericyte/myofibroblast marker). Mid‐diaphyseal defects measuring 2 mm in length were created in the central 1/3 of mice femora using a circular saw and stabilized with an alveolar distractor device and cerclage wires. Three groups were evaluated: Group I, PTH 30 μg/kg injection daily, Group II, PTH 30 μg/kg injection daily + DBM, and Group III, DBM + 30μL saline injection. PTH was given for 28 days or until the time of sacrifice. Animals were sacrificed at 7, 14, 28, and 56 days. Radiographs at the time of sacrifice were evaluated using a 5‐point scaled scoring system. Radiographs showed a lack of healing across all treatment groups at all time points: Group I, 1.57 +/? 0.68; Group II, 3.00 +/? 1.29; and Group III, 2.90 +/? 1.03. Bone formation in the defect as measured by radiographic healing score was significantly better at 56 days in Groups II (p = 0.01) and III (p < 0.01) compared to Group I. Across all treatment groups and time points the defects were largely absent of osteoprogenitor cells based on gross observation of frozen histology and quantitation of cellular based histomorphometric parameters. Quantitation of frozen histologic slides showed a limited osteoprogenitor response to PTH and DBM. Our results suggest that the anabolic agent teriparatide is unable to induce healing in a critical sized mouse femoral defect when given alone or in combination with the DBM preparation we used as a local bone graft substitute. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1242–1249, 2015.

     

Activated protein C (APC) can increase bone anabolism via a protease‐activated receptor (PAR)1/2 dependent mechanism

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP‐2) induced ectopic bone formation model. Local co‐administration of 10 µg rhBMP‐2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p < 0.01) compared to rhBMP‐2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease‐activated receptors (PARs). Cultured pre‐osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre‐osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP‐2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1549–1556, 2014.     

Col1a1-driven transgenic markers of osteoblast lineage progression.

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.     

Nanocomposite therapy as a more efficacious and less inflammatory alternative to bone morphogenetic protein‐2 in a rodent arthrodesis model

The use of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) in spine fusion has led to concerns regarding a potential accompanying inflammatory response. This study evaluates a combination therapy (TrioMatrix®; Pioneer Surgical, Inc., Marquette, MI) comprised of a demineralized bone matrix (DBM), hydroxyapatite, and a nanofiber‐based collagen scaffold in a rodent spine fusion model. Thirty‐six athymic rats that underwent a posterolateral intertransverse spinal fusion were randomly assigned to 1 of 5 treatment groups: absorbable collagen sponge alone (ACS, negative control), 10 µg rhBMP‐2 on ACS (positive control), TrioMatrix®, Grafton® (Osteotech, Inc., Eatontown, NJ), and DBX® (Synthes, Inc., West Chester, PA). Both TrioMatrix® and rhBMP‐2‐treated animals demonstrated 100% fusion rates as graded by manual palpation scores 8 weeks after implantation. This rate was significantly greater than those of the ACS, Grafton®, and DBX® groups. Notably, the use of TrioMatrix® as evaluated by microCT quantification led to a greater fusion mass volume when compared to all other groups, including the rhBMP‐2 group. T2‐weighted axial MRI images of the fusion bed demonstrated a significant host response associated with a large fluid collection with the use of rhBMP‐2; this response was significantly reduced with the use of TrioMatrix®. Our results therefore demonstrate that a nanocomposite therapy represents a promising, cost‐effective bone graft substitute that could be useful in spine fusions where BMP‐2 is contraindicated. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1812–1819, 2011     

Effect of rhBMP‐2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long‐term immunosuppression using short‐term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein‐2 (rhBMP‐2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra‐medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP‐2 or rhBMP‐2 + VEGF. FK‐506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP‐2 + VEGF) group than rhBMP‐2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP‐2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP‐2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 561–566, 2013     

An Activin A/BMP2 Chimera,AB204, Displays Bone‐Healing Properties Superior to Those of BMP2

Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10‐fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2‐like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100‐fold greater than that of BMP2. The structure also led to our identification of a single Activin A‐derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non‐union bone fractures. © 2014 American Society for Bone and Mineral Research.     

Repair of Osteochondral Defects in a Rabbit Model Using a Porous Hydroxyapatite Collagen Composite Impregnated With Bone Morphogenetic Protein‐2

Articular cartilage has a limited capacity for spontaneous repair, and an effective method to repair damaged articular cartilage has not yet been established. The purpose of this study was to evaluate the effect of transplantation of porous hydroxyapatite collagen (HAp/Col) impregnated with bone morphogenetic protein‐2 (BMP‐2). To evaluate the characteristics of porous HAp/Col as a drug delivery carrier of recombinant human BMP‐2 (rhBMP‐2), the rhBMP‐2 adsorption capacity and release kinetics of porous HAp/Col were analyzed. Porous HAp/Col impregnated with different amounts of rhBMP‐2 (0, 5, and 25 μg) was implanted into osteochondral defects generated in the patellar groove of Japanese white rabbits to evaluate the effect on osteochondral defect regeneration. At 3, 6, 12, and 24 weeks after operation, samples were harvested and subjected to micro‐computed tomography analysis and histological evaluation of articular cartilage and subchondral bone repair. The adsorption capacity was 329.4 μg of rhBMP‐2 per cm³ of porous HAp/Col. Although 36% of rhBMP‐2 was released within 24 h, more than 50% of the rhBMP‐2 was retained in the porous HAp/Col through the course of the experiment. Defects treated with 5 μg of rhBMP‐2 showed the most extensive subchondral bone repair and the highest histological regeneration score, and differences against the untreated defect group were significant. The histological regeneration score of defects treated with 25 μg of rhBMP‐2 increased up to 6 weeks after implantation, but then decreased. Porous HAp/Col, therefore, is an appropriate carrier for rhBMP‐2. Implantation of porous HAp/Col impregnated with rhBMP‐2 is effective for rigid subchondral bone repair, which is important for the repair of the smooth articular surface.     

Enhancement of recombinant human BMP‐7 bone formation with bmp binding peptide in a rodent femoral defect model

Bone morphogenetic binding peptide (BBP) is an 18.5 kDa fragment of a bone matrix protein peptide. A rat femoral defect model was used to test the effect of BBP combined with recombinant human bone morphogenetic protein‐7 (rhBMP‐7) to induced bone healing. Two doses of BBP (500 and 1000 µg) were tested with two doses of rhBMP‐7 (2 and 5 µg), and the results were compared with a positive control (10 µg rhBMP‐7). Bone healing was evaluated by radiology, manual palpation, microcomputed tomography, and histology. The high dose of 10 µg of rhBMP‐7 resulted in a consistent 100% bone union rate and a mature histological appearance on histology, and was used as a positive control. When 1000 µg of BBP was combined with lower doses of BMP‐7 (2 µg rhBMP‐7 or 5 µg rhBMP‐7) significant differences were seen in radiographic scores, manual palpation, and bone volume, when compared to 2 µg rhBMP‐7 or 5 µg rhBMP‐7 alone. The combination of 1000 µg of BBP and 5 µg rhBMP‐7 also achieved 100% fusion rate, induced a larger amount of bone formation, and yielded similar maturity of bone marrow when compared with the high dosage 10 µg rhBMP‐7 group. This study demonstrated that when combined together, BBP can enhance the bone healing of rhBMP‐7. Improved healing imparted by the addition of BBP may result in lesser amounts of rhBMP‐7 needed to achieve union in the clinical setting. © 2010 OrthopaedicResearchSociety.PublishedbyWileyPeriodicals, Inc.JOrthopRes29:753–759,2011     

Augmentation of fracture healing by hydroxyapatite/collagen paste and bone morphogenetic protein‐2 evaluated using a rat femur osteotomy model

In fracture treatment, biological bone union generally depends on the bone's natural fracture healing capacity, even in surgically treated cases. Hydroxyapatite/collagen composite (HAp/Col) has high osteoconductivity and stimulates osteogenic progenitors. Furthermore, it has the potent capacity to adsorb bone morphogenetic proteins (BMPs). In this study, we prepared an injectable HAp/Col paste and evaluated its augmentation of bone union. Furthermore, the effect of HAp/Col paste combined with BMP‐2 was also evaluated. We used a rat femur osteotomy model with a defect size of 1 mm. Male Wistar rats were assigned to one of the following four groups; a control group without any implant, a HAp/Col implant group, a group that received an absorbable collagen sponge (ACS) implant impregnated with BMP‐2 (1 μg), and a group that received a HAp/Col implant impregnated with BMP‐2 implant. Micro‐CT analysis, three‐point bending tests, and histological evaluation were performed. Bone union was achieved in two of eight cases in the HAp/Col group, five of eight cases in the ACS + BMP‐2 group, and all cases in the HAp/Col + BMP‐2 group at 8 weeks post‐surgery. The control group did not achieve bone union. In addition, in the HAp/Col + BMP‐2 group, the biomechanical strength of the fused femurs was comparable to that of the contralateral intact femur; the ratio of the mechanical load at the breaking point of the osteotomy side relative to that of the contralateral side was 1.00 ± 0.151 (SD). These results indicate that HAp/Col paste with or without BMP‐2 augments bone union. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:129–137, 2018.     

Influence of the ratio of particulate autogenous bone graft/platelet‐rich plasma on bone healing in critical‐size defects: A histologic and histometric study in rat calvaria

The purpose of this study was to analyze histomorphometrically the influence of the ratio of particulate autogenous bone (AB) graft/platelet‐rich plasma (PRP) on bone healing in surgically created critical‐size defects (CSD) in rat calvaria. Fifty rats were divided into five groups: Group C (control), Group AB, Group AB/PRP‐50, Group AB/PRP‐100, and Group AB/PRP‐150. A 5‐mm diameter critical‐size defect was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group AB, the defect was filled with 0.01 mL of AB graft. In Groups AB/PRP‐50, AB/PRP‐100, and AB/PRP‐150, the defects were filled with 0.01 mL of AB graft combined with 50, 100, and 150 µL of PRP, respectively. All animals were euthanized at 30 days postoperative. Histomorphometry, using image analysis software, and histology analyses were performed. New Bone Area (NBA) and the remaining bone graft particles area (RPA) were calculated as a percentage of the total area of the original defect. Percentage data were transformed into arccosine for analysis. No defect completely regenerated with bone. Group AB/PRP‐50 (41.78 ± 13.48%) had a significantly greater NBA than Groups C (19.29 ± 5.11%), AB (27.43 ± 10.90%) or AB/PRP‐150 (19.17 ± 8.45%) (p < 0.05). No significant differences were observed between groups AB/PRP‐50 and AB/PRP‐100 or among groups AB, AB/PRP‐100, and AB/PRP‐150 with regard to NBA (p > 0.05). Group AB/PRP‐150 (31.59 ± 3.22%) had a significantly greater RPA than Groups AB (19.09 ± 5.21%), AB/PRP‐50 (17.33 ± 4.43%), and AB/PRP‐100 (19.72 ± 3.62%) (p < 0.001). No significant differences were observed among groups AB, AB/PRP‐50, and AB/PRP‐100 with regard to RPA (p > 0.05). The ratio AB graft/PRP influences bone healing in surgically created CSD in rat calvaria. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:468–473, 2010     

Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage.

Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.     

Controlled‐release of bone morphogenetic protein‐2 from a microsphere coating applied to acid‐etched Ti6AL4V implants increases biological bone growth in vivo

A central clinical challenge regarding the surgical treatment of bone and joint conditions is the eventual loosening of an orthopedic implant as a result of insufficient bone ingrowth at the bone–implant interface. We investigated the in vivo effectiveness of a coating containing recombinant human bone morphogenetic protein‐2 (rhBMP‐2)‐loaded microspheres applied to acid‐etched Ti6Al4V cylinders for implantation. Three groups of rabbits (24 per group) were used for implantation: (1) acid‐etched Ti6Al4V implants coated with a mixture of rhBMP‐2‐loaded microspheres (125 ng rhBMP‐2/mg microspheres) and α‐butyl cyanoacrylate; (2) acid‐etched, uncoated implants; and (3) bare, smooth uncoated implants. After implantation, 12 rabbits from each group were used for bone ingrowth determination at 4, 5, 6, 7, 8, and 12 weeks (2 rabbits per time point), while the remainder were used for histological analysis and push‐out testing at 12 weeks. Scanning electron microscopy showed significant improvement in bone growth of the rhBMP‐2 microspheres/α‐butyl cyanoacrylate group compared with the other groups (p < 0.01). Histological analysis and push‐out testing also demonstrated enhanced bone growth of the rhBMP‐2 group over that in the other two groups (p < 0.01). The rhBMP‐2 group showed the most significant bone growth, suggesting that coating acid‐etched implants with a mixture of rhBMP‐2‐loaded microspheres and α‐butyl cyanoacrylate may be an effective method to improve the osseointegration of orthopedic implants. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:744–751, 2014.     

Osteoblast‐targeted overexpression of PPARγ inhibited bone mass gain in male mice and accelerated ovariectomy‐induced bone loss in female mice

PPARγ has critical role in the differentiation of mesenchymal stem cells into adipocytes while suppressing osteoblastic differentiation. We generated transgenic mice that overexpress PPARγ specifically in osteoblasts under the control of a 2.3‐kb procollagen type 1 promoter (Col.1‐PPARγ). Bone mineral density (BMD) of 6‐ to 14‐week‐old Col.1 ? PPARγ male mice was 8% to 10% lower than that of their wild‐type littermates, whereas no difference was noticed in Col.1‐PPARγ female mice. Col.1‐PPARγ male mice exhibited decreased bone volume (45%), trabecular thickness (23%), and trabecular number (27%), with a reciprocal increase in trabecular spacing (51%). Dynamic histomorphometric analysis also revealed that bone‐formation rate (42%) and mineral apposition rate (32%) were suppressed significantly in Col.1‐PPARγ male mice compared with their wild‐type littermates. Interestingly, osteoclast number and surface also were decreased by 40% and 58%, respectively, in Col.1‐PPARγ male mice. In vitro whole‐marrow culture for osteoclastogenesis also showed a significant decrease in osteoclast formation (approximately 35%) with the cells from Col.1‐PPARγ male mice, and OPG/RANKL ratio was reduced in stromal cells from Col.1‐PPARγ male mice. Although there was no significant difference in BMD in Col.1‐PPARγ female mice up to 30 weeks, bone loss was accelerated after ovariectomy compared with wild‐type female mice (?3.9% versus ?6.8% at 12 weeks after ovariectomy, p < .01), indicating that the effects of PPARγ overexpression becomes more evident in an estrogen‐deprived state in female mice. In conclusion, in vivo osteoblast‐specific overexpression of PPARγ negatively regulates bone mass in male mice and accelerates estrogen‐deficiency‐related bone loss in female mice. © 2011 American Society for Bone and Mineral Research     

Imaging early stage osteogenic differentiation of mesenchymal stem cells

Stem cells, such as mesenchymal stem cells (MSCs), contribute to bone fracture repair if they are delivered to the injury site. However, it is difficult to assess the retention and differentiation of these cells after implantation. Current options for non‐invasively tracking the transplanted stem cells are limited. Cell‐based therapies using MSCs would benefit greatly through the use of an imaging methodology that allows cells to be tracked in vivo and in a timely fashion. In this study, we implemented an in vivo imaging methodology to specifically track early events such as differentiation of implanted human MSCs (hMSCs). This system uses the collagen type 1 (Col1α1) promoter to drive expression of firefly luciferase (luc) in addition to a constitutively active promoter to drive the expression of green fluorescent protein (GFP). The resulting dual‐promoter reporter gene system provides the opportunity for osteogenic differentiation‐specific luc expression for in vivo imaging and constitutive expression of GFP for cell sorting. The function of this dual‐promoter reporter gene was validated both in vitro and in vivo. In addition, the ability of this dual‐promoter reporter system to image an early event of osteogenic differentiation of hMSCs was demonstrated in a murine segmental bone defect model in which reporter‐labeled hMSCs were seeded into an alginate hydrogel scaffold and implanted directly into the defect. Bioluminescence imaging (BLI) was performed to visualize the turn‐on of Col1α1 upon osteogenic differentiation and followed by X‐ray imaging to assess the healing process for correlation with histological analyses. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res XX:XXX–XXX, 2013 © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 871–879, 2013     

Selective laser melting‐produced porous titanium scaffolds regenerate bone in critical size cortical bone defects

Porous titanium scaffolds have good mechanical properties that make them an interesting bone substitute material for large bone defects. These scaffolds can be produced with selective laser melting, which has the advantage of tailoring the structure's architecture. Reducing the strut size reduces the stiffness of the structure and may have a positive effect on bone formation. Two scaffolds with struts of 120‐µm (titanium‐120) or 230‐µm (titanium‐230) were studied in a load‐bearing critical femoral bone defect in rats. The defect was stabilized with an internal plate and treated with titanium‐120, titanium‐230, or left empty. In vivo micro‐CT scans at 4, 8, and 12 weeks showed more bone in the defects treated with scaffolds. Finally, 18.4 ± 7.1 mm³ (titanium‐120, p = 0.015) and 18.7 ± 8.0 mm³ (titanium‐230, p = 0.012) of bone was formed in those defects, significantly more than in the empty defects (5.8 ± 5.1 mm³). Bending tests on the excised femurs after 12 weeks showed that the fusion strength reached 62% (titanium‐120) and 45% (titanium‐230) of the intact contralateral femurs, but there was no significant difference between the two scaffolds. This study showed that in addition to adequate mechanical support, porous titanium scaffolds facilitate bone formation, which results in high mechanical integrity of the treated large bone defects. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 792–799, 2013     

Mesenchymal stem cells accelerate bone allograft incorporation in the presence of diabetes mellitus

Allograft (Allo) incorporation in the presence of a systemic disease like diabetes mellitus (DM) is becoming a major issue in the orthopedic community. Mesenchymal stem cells (MSC) are multipotent stem cells that may be derived from adult, whole bone marrow and have been shown to induce bone formation in segmental defects when combined with the appropriate carrier/scaffold. The objectives of this study were to analyze the effect of DM upon Allo incorporation in a segmental rat femoral defect and to also investigate MSC augmentation of Allo incorporation. Segmental (5 mm) femoral defects were created in non‐DM and DM rats and treated with Allo containing demineralized bone matrix (DBM) or DBM with MSC augmentation. Histological scoring at 4 weeks demonstrated less mature bone in the DM/DBM group compared to its non‐DM counterpart (p < 0.001). However, there was significantly more mature bone in the DM/MSC group when compared to the DM/DBM group at both 4 and 8 weeks (p < 0.001 and p = 0.004). Furthermore, significantly more bone formation was observed in the DM/MSC group compared to the DM/DBM group at the 4‐week time point (p < 0.001). The results of this study suggest that MSC are a potential adjunct for bone regeneration when implanted in an orthotopic site in the presence of DM. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:942–949, 2010     

Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model

Purpose

Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model.

Methods

Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks.

Results

We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells.

Conclusion

We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.     

Evaluation of the effects of systemic treatment with a sclerostin neutralizing antibody on bone repair in a rat femoral defect model

Systemic administration of a sclerostin neutralizing antibody (Scl‐Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl‐Ab to improve healing in a bone defect model, we evaluated Scl‐Ab in a 3 mm femoral defect in young male outbred rats. Scl‐Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, µ‐CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl‐Ab treated 5/30, vehicle treated 1/15). µ‐CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p < 0.05), but a non‐significant increase in the continuous group (35%, p = 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un‐operated femurs. We demonstrate that treatment with Scl‐Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:197–203, 2014.