Healing of an ulnar defect using a proprietary TCP bone graft substitute, JAX, in association with autologous osteogenic cells and growth factors

Currently, available synthetic bone substitutes have adequate osteoconductive properties but have little or no osteoinductivity. Recent research has focused on using osteogenic growth factors or cells to provide this. JAX is a beta tricalcium phosphate bone graft substitute that has a novel shape and interlocking design. This study investigated delivery methods and the use of autologous cell therapy to enhance healing of a bone defect using JAX as a scaffold. Bone marrow was harvested from 24 New Zealand White rabbits. The mononuclear cell fraction was isolated and culture expanded. Bilateral 1.5 cm defects in the ulna were filled with: Group 1: JAX alone, Group 2: JAX plus 1x10(7) autologous BMSCs injected at the time of surgery, Group 3: JAX plus 8x10(6) autologous BMSCs cultured on granules for 14 days prior to surgery, Group 4: JAX plus fresh bone marrow (BMA), Group 5: cortical autograft, Group 6: JAX plus 2.5 microg VEGF. Radiographs demonstrated that there was more new bone in the BMA and VEGF groups compared to JAX alone. Groups containing autologous BMSCs were only slightly better than JAX alone in the amount of bone in the defect but did improve bridging of the osteotomy. Histomorphometry identified a significant increase in bone volume in the BMA group compared to JAX alone. BMA and VEGF enhanced healing of bone defects whereas expanded BMSCs provided little advantage over scaffold alone. There was no difference between delivery methods of autologous BMSCs. These observations suggest that the provision of osteogenic cells alone is insufficient to enhance bone healing and that additional factors are required to initiate this process in vivo.     

IL‐7 overexpression enhances therapeutic potential of rat bone marrow mesenchymal stem cells for diabetic wounds

This study was aimed to enhance the healing potential of rat bone marrow mesenchymal stem cells against chronic diabetic wounds through interleukin‐7 (IL‐7) transfection. IL‐7 plays an important role in wound healing and acts as a survival factor in some cell types. This study involves isolation, propagation, and characterization of mesenchymal stem cells (MSCs) and their modification with IL‐7 gene via retroviral transfection. Transfected MSCs were assessed for their effect on angiogenic genes by qPCR. Wound healing potential of transfected MSCs was analyzed by scratch assay in vitro and by transplanting these cells in rat diabetic wound models in vivo. Wound area was measured for a period of 15 days and subsequent histological analysis was performed. qPCR results showed increased expression of IL‐7 gene (p ≤ 0.05) and also principal angiogenic genes, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), VEGF receptor 1 (FLT‐1), and VEGF receptor 2 (FLK‐1) (p ≤ 0.05). Neuropilin‐1 (NRP‐1) did not show any significant change. In vitro analysis of IL‐7 MSCs showed intense cell–cell connections and tube formation as compared to the normal MSCs. Rate of wound closure was more (p ≤ 0.001) in case of diabetic group transplanted with IL‐7 MSCs. Histological examination revealed enhanced vascular supply in skin tissues of diabetic animals transplanted with IL‐7 transfected MSCs as compared to normal MSCs. Immunohistochemical results showed significantly higher expression of IL‐7 (p ≤ 0.001) and α‐smooth muscle actin(p ≤ 0.001) in the tissue sections of IL‐7 transfected group as compared to normal MSCs and the diabetic control group; the latter indicates increase in the number of blood vessels. It is concluded from this study that IL‐7 overexpression in MSCs can enhance the healing potential of MSCs and aid in wound closure in diabetic animals through the induction of angiogenic genes.     

Effect of Panax ginseng extract on the activity of diabetic fibroblasts in vitro

Numerous studies have demonstrated the various medicinal properties of Panax ginseng, including angiogenic, immuno‐stimulating, antimicrobial, and anti‐inflammatory activities, which can be helpful in chronic wound healing. However, a direct role for P. ginseng in chronic wound healing has not been demonstrated. The present study was designed to evaluate the effects of P. ginseng extract on diabetic fibroblasts in vitro. Human diabetic fibroblasts were cultured in the presence of Ginsenoside Rb1 (G‐Rb1), the active component in P. ginseng (10 ng/mL), and untreated diabetic fibroblasts were used as controls. Cell proliferation, collagen synthesis, the production of various growth factors (basic fibroblast growth factor [bFGF]; vascular endothelial growth factor [VEGF]; and transforming growth factor‐β1 [TGF‐β1]), and the synthesis of matrix metalloproteinase 1 (MMP‐1) and tissue inhibitor of metalloproteinases 1 (TIMP‐1) were compared using enzyme‐linked immunosorbent assay and immunofluorescence staining. Compared with the control group, G‐Rb1‐treated fibroblasts showed significantly (P < 0.05) higher levels of cell proliferation, collagen synthesis, VEGF, TGF‐β1, and TIMP‐1. However, no significant differences in bFGF and MMP‐1 levels were observed between the two groups. These results suggest that P. ginseng treatment may stimulate the wound‐healing activity of diabetic fibroblasts in vitro.     

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.

     

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     

Manipulation of the anabolic and catabolic responses with BMP-2 and zoledronic acid in a rat femoral fracture model

Bone repair involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the fracture site. It has been reported that the optimal time point for single-dose zoledronic acid (ZA) administration systemically increased the strength of bone morphogenetic protein (BMP)-7-mediated callus. However, its repair mechanism during bone fracture healing remains unknown. We aimed to investigate the synergic effect of recombinant human (rh) BMP-2 and ZA in a rat femoral fracture model. Fifty-eight rats were divided into 4 groups. Group I (n = 14) animals were implanted with a carrier alone. Group II (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2. Group III (n = 14) animals were implanted with a carrier and a subcutaneous systemic ZA injection 2 weeks after surgery. Group IV (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2 and ZA subcutaneous injection 2 weeks after surgery. The rats were euthanized after 6 weeks and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high-resolution micro-computerized tomography (micro-CT) and were subjected to biomechanical and histological analysis. The fusion rates in Group IV (93.3%) were considerably higher than those in Groups I (28.6%), II (53.3%), and III (57.1%). Additionally, the radiographic scores of Group IV were higher than those in Groups I, II, and III. In micro-CT analysis, the tissue volume (TV) of the callus was higher in Group IV than in Groups I and II (p < 0.05). New bone volume (BV) and trabecular spacing (Tb.Sp) also showed essentially the same trend as that of TV. The ratio of BV to TV (BV/TV), the trabecular number (Tb.N), and the trabecular thickness (Tb.Th) was higher in Groups III and IV than in Groups I and II (p < 0.05). In biomechanical analysis, the ultimate loads at failure and stiffness in Groups III and IV were on average higher than those in Groups I and II (p < 0.05), while the energy absorption of Group IV was higher than those of Groups I and II (p < 0.05). The synergic effect of rhBMP-2 and ZA given systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion. Our results suggest that this combination facilitates bone healing and has potential clinical application.     

Neurotrophin‐3 Induces BMP‐2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill‐hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT‐3, and NT‐4 and their Trk receptors. NT‐3 and its receptor TrkC showed the highest induction. NT‐3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT‐3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT‐3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP‐2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT‐3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP‐2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT‐3 osteogenic effect in vitro because it can be almost completely abrogated by co‐addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT‐3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co‐treatment with anti‐VEGF. This study suggests that NT‐3 may be an osteogenic and angiogenic factor upstream of BMP‐2 and VEGF in bony repair, and further studies are required to investigate whether NT‐3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.     

Sildenafil accelerates fracture healing in mice

Sildenafil, a cyclic guanosine monophosphate (cGMP)‐dependent phospodiesterase‐5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro‐angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro‐angiogenic and osteogenic cysteine‐rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61‐associated pathway. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:867–873     

Role and regulation of VEGF and its receptors 1 and 2 in the aseptic loosening of total hip implants

It was hypothesized that vascular endothelial growth factor (VEGF) in fibroblasts participates in aseptic loosening of total hip replacement (THR) implants. Therefore, osteoarthritic (OA) samples (n = 11) were compared with synovial membrane‐like interface tissues from revision THR (n = 10). VEGF‐A and its receptors were stained using streptavidin‐immunoperoxidase method. Their regulation by hypoxia and cytokines were studied in cultured fibroblasts using quantitative real‐time polymerase chain reaction (qRT‐PCR). VEGFR1⁺ lining cells (p < 0.01), stromal fibroblast‐like cells (p = 0.001) and stromal macrophage‐like cells (p < 0.05) were more numerous in rTHR than in OA. As to VEGFR2⁺, only stromal fibroblast‐like cells in rTHR outnumbered those found in OA (p < 0.05). VEGFRs in synovial fibroblasts were not affected by hypoxia, but VEGF increased 2.4‐fold (p < 0.05). Interleukin‐4 up‐regulated VEGFR1 expression 23‐fold. This is the first study to describe a difference between rTHR and OA in VEGF receptors, particularly VEGFR1. Hypoxia increased VEGF, but the VEGFR1 increase in the lining and stroma is probably IL‐4 driven, in accordance with the M2‐type macrophage dominance in interface tissues. VEGF/VEGFR system is also affected by hypoxia and may play a role in angiogenesis and bone pathology in aseptic loosening of total hip implants. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1830–1836, 2012     

Hypoandrogenism related to early skin wound healing resistance in rats

The purpose of this study was to verify the effect of testosterone depletion on healing of surgical skin wounds at different ages and post‐operative periods. Forty‐four Wistar male rats were divided into four groups: Group 1Y (n = 11) – young control, sham‐operated rats (30‐day old); Group 1A (n = 10) – adult control, sham‐operated rats (3 to 4‐month old); Group 2Y (n = 10) – young rats after bilateral orchiectomy; and Group 2A (n = 11) – adult rats after bilateral orchiectomy. After 6 months, a linear incision was performed on the dorsal region of the animals. The resistance of the wound healing was measured in a skin fragment using a tensiometer, on the 7th and 21st post‐operative days. The wound healing resistance was higher in Group 1Y than in Group 2Y after 7 days (P < 0.05). Wound healing resistance at 21 days was higher than at 7 days in all groups (P < 0.05). Late wound healing resistance was not different between young and adult rats. It is concluded that bilateral orchiectomy diminished the wound healing resistance only in young animals at the 7th post‐operative day.     

Exercise enhances angiogenesis during bone defect healing in mice

The aim of the present study was to investigate the effect of exercise on angiogenesis during bone defect healing in mice. We evaluated angiogenesis during cranial bone defect healing by intravital fluorescence microscopy (IVM) at days 0–21. To characterize the type of bone repair, we performed additional histomorphometric analyses at days 3–15. IVM was conducted in mice, which were housed in cages supplied with running wheels (exercise group; n = 7) and compared to IVM results of mice, which were housed in cages without running wheels (controls; n = 7). In the exercise group, we additionally performed correlation analyses between results of the IVM and the running distance. IVM showed an accelerated decrease of bone defect area in the exercise group compared to the control group. This was associated with a significantly higher blood vessel diameter in animals undergoing exercise at days 9 and 12 and a significant correlation between running distance and blood vessel density at day 9 (r = 0.74). Histomorphometry showed osseous bridging of the defect at day 9. The newly woven bone was covered by a neo‐periosteum containing those blood vessels, which were visible by IVM. We conclude that exercise accelerates bone defect healing and stimulates angiogenesis during bore repair. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1086–1092, 2011     

Strut-autografting with and without osteogenic protein-1 : a preliminary study of a canine femoral head defect model.

BACKGROUND: Osteonecrosis of the femoral head frequently leads to collapse of the articular cartilage and to disabling osteoarthritis, which ultimately may necessitate joint arthroplasty. One treatment method that has had moderate success is the so-called trapdoor approach, which involves excavation of diseased (necrotic) bone followed by bone-grafting. Augmentation of this procedure with various growth and differentiation factors may improve the outcome. We developed a canine model that mimics the clinical situation with trapdoor bone-grafting. The objective of this study was to evaluate the effect of the addition of osteogenic protein-1 on healing following the trapdoor procedure with strut-autografting. METHODS: Thirty-four skeletally mature dogs were used in the experiment. After capsulotomy, a trapdoor was created in the anterolateral surface of the femoral head and a 2-cm-diameter subchondral area of bone was removed. In the phase-I experiments, seven dogs had no treatment of the defect (Group I) and nine dogs were treated with strut-grafting (Group II). In phase II, the procedure was modified by collapsing the trapdoor into the created defect intraoperatively in eighteen dogs, which were divided into three equal groups: six untreated defects were left collapsed (Group III), six were treated with bone graft (Group IV), and six were treated with bone graft augmented with osteogenic protein-1 (Group V). RESULTS: Three of the seven femoral heads in Group I (untreated defect) and one of the nine heads in Group II (grafting without collapsing of the trapdoor) had evidence of cartilage collapse. Inspection of sagittal slices and radiographs revealed an unfilled residual defect in all Group-I heads, whereas all Group-II heads were well healed. The mean normalized stiffness value was significantly larger in Group II than it was in Group I. On visual inspection, depression was noted in all of the femoral heads in Group III (untreated defect; trapdoor left collapsed). In both Group IV and Group V (grafting without and with osteogenic protein-1), the trapdoor cartilage appeared to be essentially normal. Groups IV and V had more radiographic healing than did Group III. The defects in Group V (grafting with osteogenic protein-1) healed faster radiographically than did those in Group IV (grafting without osteogenic protein-1). CONCLUSIONS: Moderate-to-excellent healing was seen both radiographically and biomechanically by four months in the groups treated with grafting, with and without osteogenic protein-1, whereas untreated defects did not heal. Clinical Relevance: Symptomatic osteonecrosis of the femoral head is a clinical challenge. The animal model in the current study is a useful tool for the evaluation of methods to treat osteonecrosis of the femoral head. Studies investigating additional time-periods between implantation of osteogenic protein-1 and assessment of results as well as different doses of osteogenic protein-1 are warranted.     

Repair of Critical‐Sized Long Bone Defects Using Dipyridamole‐Augmented 3D‐Printed Bioactive Ceramic Scaffolds

There are over two million long bone defects treated in the United States annually, of which ~5% will not heal without significant surgical intervention. While autogenous grafting is the standard of care in simple defects, a customized scaffold for large defects in unlimited quantities is not available. Recently, a three‐dimensionally (3D)‐printed bioactive ceramic (3DPBC) scaffold has been successfully utilized in the of repair critical‐sized (CSD) long bone defects in vivo. In this study, 3DPBC scaffolds were augmented with dipyridamole (DIPY), an adenosine A2A receptor (A_2AR) indirect agonist, because of its known effect to enhance bone formation. CSD full thickness segmental defects (~11 mm × full thickness) defects were created in the radial diaphysis in New Zealand white rabbits (n = 24). A customized 3DPBC scaffold composed of β‐tricalcium phosphate was placed into the defect site. Groups included scaffolds that were collagen‐coated (COLL), or immersed in 10, 100, or 1,000 μM DIPY solution. Animals were euthanized 8 weeks post‐operatively and the radii/ulna‐scaffold complex retrieved en bloc, for micro‐CT, histological, and mechanical analysis. Bone growth was assessed exclusively within scaffold pores and evaluated by microCT and advanced reconstruction software. Biomechanical properties were evaluated utilizing nanoindentation to assess the newly regenerated bone for elastic modulus (E) and hardness (H). MicroCT reconstructions illustrated bone in‐growth throughout the scaffold, with an increase in bone volume dependent on the DIPY dosage. The histological evaluation did not indicate any adverse immune response while revealing progressive remodeling of bone. These customized biologic 3DPBC scaffolds have the potential of repairing and regenerating bone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2499–2507, 2019     

Enhancement of the Effects of Exfoliated Carbon Nanofibers by Bone Morphogenetic Protein in a Rat Femoral Fracture Model

Exfoliated carbon nanofibers (ExCNFs) are expected to serve as excellent scaffolds for promoting and guiding bone‐tissue regeneration. We aimed to enhance the effects of ExCNFs with bone morphogenetic proteins (BMPs) and examine their feasibility and safety in clinical applications using a rat femoral fracture model. Group I (n = 16) animals were implanted with control MedGEL. Group II (n = 17) animals were implanted with MedGEL containing ExCNFs. Group III (n = 15) animals were implanted with MedGEL containing 1 μg rhBMP‐2. Group IV (n = 15) animals were implanted with MedGEL containing 1 μg rhBMP‐2 and ExCNFs. The rats were euthanized after 6 weeks, and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high‐resolution microcomputerized tomography (micro‐CT); the femurs were also subjected to biomechanical and histological analysis. The fusion rates in Group IV (73.3%) were considerably higher than those in Groups I (25.0%), II (52.9%), and III (46.7%). The results demonstrated the enhancement of the bone repair effects of ExCNFs by BMP in a rat femoral fracture model. Our results suggest that the enhancement of the effects of ExCNFs by BMP makes the combination a possible attractive therapy for various orthopedic surgeries. © 2014 Orthopaedic Research Society. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:185–192, 2015.

     

Soluble VEGFR1 reverses BMP2 inhibition of intramembranous ossification during healing of cortical bone defects

BMP2 is widely used for promotion of bone repair and regeneration. However, bone formation induced by BMP2 is quite variable. Bone forming progenitor cells in different locations appear to respond to BMP2 in different ways, and repair outcomes can vary as a consequence of modulating effects by other factors. In this study, we have examined the effects of VEGF on BMP2‐induced repair of a cortical bone defect, a 1 mm diameter drill hole, in the proximal tibia of mice. Treatment of the defect with either a bolus of PBS or soluble VEGFR1 (sVEGFR1), a decoy receptor for VEGF, had the same effects on bone formation via intramembranous ossification in the defect and cartilage formation and injured periosteum, during the healing process. In contrast, treatment with BMP2 inhibited intramembranous bone formation in the defect while it promoted cartilage and endochondral bone formation in the injured periosteum compared with mice treated with PBS or sVEGFR1. The inhibitory effect of BMP2 on bone formation was unlikely due to increased osteoclast activity and decreased invasion of blood vessels in the defect. Most importantly, co‐delivery of BMP2 and sVEGFR1 reversed the inhibition of intramembranous bone formation by BMP2. Furthermore, the decreased accumulation of collagen and production of bone matrix proteins in the defect of groups with BMP2 treatment could also be prevented by co‐delivery of BMP2 and sVEGFR1. Our data indicate that introducing a VEGF‐binding protein, such as sVEGFR1, to reduce levels of extracellular VEGF, may enhance the effects of BMP2 on intramembranous bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1461–1469, 2017.

     

Induction of angiogenesis and osteogenesis in surgically revascularized frozen bone allografts by sustained delivery of FGF‐2 and VEGF

Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF‐2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF‐2, VEGF, or FGF‐2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF‐2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF‐2 + VEGF was observed. VEGF delivered in microspheres had superior long‐term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF‐2 or FGF‐2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1556–1562, 2012     

Polydeoxyribonucleotide stimulates angiogenesis and wound healing in the genetically diabetic mouse

Healing of diabetic wounds still remains a critical medical problem. Polydeoxyribonucleotide (PDRN), a compound having a mixture of deoxyribonucleotide polymers, stimulates the A2 purinergic receptor with no toxic or adverse effect. We studied the effects of PDRN in diabetes‐related healing defect using an incisional skin‐wound model produced on the back of female diabetic mice (db+/db+) and their normal littermates (db+/+m). Animals were treated daily for 12 days with PDRN (8 mg/kg/ip) or its vehicle (100 μL 0.9%NaCl). Mice were killed 3, 6, and 12 days after skin injury to measure vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis, to assay angiogenesis and tissue remodeling through histological evaluation, and to study CD31, Angiopoietin‐1 and Transglutaminase‐II. Furthermore, we measured wound breaking strength at day 12. PDRN injection in diabetic mice resulted in an increased VEGF message (vehicle=1.0±0.2 n‐fold vs. β‐actin; PDRN=1.5±0.09 n‐fold vs. β‐actin) and protein wound content on day 6 (vehicle=0.3±0.07 pg/wound; PDRN=0.9±0.1 pg/wound). PDRN injection improved the impaired wound healing and increased the wound‐breaking strength in diabetic mice. PDRN also caused a marked increase in CD31 immunostaining and induced Transglutaminase‐II and Angiopoietin‐1 expression. Furthermore, the concomitant administration of 3,7‐dimethyl‐1‐propargilxanthine, a selective adenosine A2A receptor antagonist, abolished PDRN positive effects on healing. However, 3,7‐dimethyl‐1‐propargilxanthine alone did not affect wound healing in both diabetic mice and normal littermates. These results suggest that PDRN might be useful in wound disorders associated with diabetes.     

Long-term angiogenic activity of free grafts and pedicle flap in a rabbit urethroplasty model

Purpose

We studied the late angiogenic activity of free grafts and a pedicle flap in a rabbit urethroplasty model to determine whether angiogenic activity plays a role in late outcomes of urethral reconstruction in rabbits.

Methods

Twenty-eight rabbits were randomly divided into five groups according to the method used to bridge a urethral defect as an onlay patch: Control, simple closure of urethral defect (Group O₁); free penile skin graft (FPSG, Group A₁); buccal mucosal graft (BuMG, Group B₁); bladder mucosal graft (BlMG, Group C₁); and pedicle penile skin flap (PPSF, Group D₁). Angiogenic activity of the patch on postoperative day 84 was assessed by immunohistochemistry.

Results

The angiogenic activity in Groups O₁, A₁, B₁, C₁, and D₁ was 23.33 ± 4.92 (means ± SD), 42.89 ± 6.52, 55.78 ± 3.46, 53.61 ± 6.17, and 24.11 ± 9.07 vessels per optical field, respectively. There were statistically significant differences (p < .001) between Group O₁ and A₁ B₁, C₁, Group A₁ and B₁, C₁, D₁, Groups B₁ and D₁ and Groups C₁ and D₁, but not between Groups O₁ and D₁ (p = 1.000) and Groups B₁ and C₁ (p = .872). The long-term angiogenic activity of all the groups was significantly lower (p < .001) than in the corresponding early groups.

Conclusions

Although the angiogenic activity of all the groups decreased in the late assessment, the buccal mucosal graft continued to exhibit elevated angiogenesis above bladder or skin (free or pedicle) graft. Therefore, buccal mucosal patch graft might be preferable because of its easier harvesting.     

Biomechanical comparison of suture anchor versus margin convergence plus suture anchor for rotator cuff repair

Objective: To evaluate results of margin convergence versus suture anchors in rotator cuff repair, and to determine which method is mechanically superior. Methods: Eighteen kangaroo shoulders were randomly divided into three groups (n = 6). A full thickness tendon defect 1.0 cm × 1.5 cm in size was created in the supraspinatus tendon at humeral insertion, simulating a massive rotator cuff tear. Three different techniques were employed for rotator cuff repair: (i) Mitek GII suture anchor alone (Group 1); (ii) margin convergence alone (Group 2); and (iii) margin convergence plus Mitek GII suture anchor (Group 3). Combined loads were applied to each specimen. After completion of cyclic loading, the construct was loaded to failure. ANOVA and LSD (Least Significant Difference) multiple comparisons of the means were applied to results. Results: Cyclic load testing showed progressive gap formation in each repaired specimen with increasing cycles. Group 1 reached 50% failure at an average of 34 cycles, Group 2 at 75 cycles and Group 3 at 73 cycles. There were significant difference between Groups 1 and 2, and Groups 1 and 3 (P ≤ 0.001). After 100 loading cycles, the average gap size was 6.8 mm, 6.1 mm and 4.7 mm in Groups 1, 2 and 3, respectively. There was a significant difference between Groups 1 and 3 (P ≤ 0.015). All specimens eventually reached failure. Conclusion: Rotator cuff repairs with margin convergence +/? suture anchor were far stronger than suture anchor alone, both in gap formation and ultimate failure load. However, progressive gap formation with cyclic loading seems inevitable after cuff repair, which may facilitate clinical understanding of the phenomena of re‐tear or residual defect.     

Augmentation of surgical angiogenesis in vascularized bone allotransplants with host‐derived a/v bundle implantation,fibroblast growth factor‐2, and vascular endothelial growth factor administration

We have previously shown experimental transplantation of living allogeneic bone to be feasible without long‐term immunosuppression by development of a recipient‐derived neoangiogenic circulation within bone. In this study, we examine the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(D,L‐lactide‐co‐glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both, were inserted intramedullarly along with a recipient‐derived arteriovenous (a/v) bundle. FK‐506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic, and histomorphometric analyses were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p < 0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p < 0.005). Bone formation rates were greater with VEGF (p < 0.01) and FGF+VEGF (p < 0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation, and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1015–1021, 2010