Novel Growth Factor Combination for Improving Rotator Cuff Repair: A Rat In Vivo Study.

BACKGROUND: The lack of healing at the repaired tendon-bone interface is an important cause of failure after rotator cuff repair. While augmentation with growth factors (GFs) has demonstrated promise, the ideal combination must target all 3 tissue types at the tendon-bone interface. HYPOTHESIS: The GF combination of transforming growth factor beta 1, Insulin-like growth factor 1, and parathyroid hormone will promote tenocyte proliferation and differentiation and improve the biomechanical and histological quality of the repaired tendon-bone interface. STUDY DESIGN: Controlled laboratory study. METHODS: In vitro, human tenocytes were cultured in the presence of the GF combination for 72 hours, and cell growth assays and the expression of genes specific to tendon, cartilage, and bone were analyzed. In vivo, adult rats (N = 46) underwent detachment and repair of the left supraspinatus tendon. A PVA-tyramine gel was used to deliver the GF combination to the tendon-bone interface. Histological, biomechanical, and RNA microarray analysis was performed at 6 and 12 weeks after surgery. Immunohistochemistry for type II and X collagen was performed at 12 weeks. RESULTS: When treated with the GF combination in vitro, human tenocytes proliferated 1.5 times more than control (P = .04). The expression of scleraxis increased 65-fold (P = .013). The expression of Sox-9 (P = .011), type I collagen (P = .021), fibromodulin (P = .0075), and biglycan (P = .010) was also significantly increased, while the expression of PPARγ was decreased (P = .007). At 6 and 12 weeks postoperatively, the quality of healing on histology was significantly higher in the GF group, with the formation of a more mature tendon-bone interface, as confirmed by immunohistochemistry for type II and X collagen. The GF group achieved a load at failure and Young modulus >1.5 times higher at both time points. Microarrays at 6 weeks demonstrated upregulation of genes involved in leukocyte aggregation (S100A8, S100A9) and tissue mineralization (Bglap, serglycin, Fam20c). CONCLUSION: The GF combination promoted protendon and cartilage responses in human tenocytes in vitro; it also improved the histological appearance and mechanical properties of the repair in vivo. Microarrays of the tendon-bone interface identified inflammatory and mineralization pathways affected by the GF combination, providing novel therapeutic targets for further research. CLINICAL RELEVANCE: The use of this GF combination is translatable to patients and may improve healing after rotator cuff repair.

Sustained Delivery of Lactoferrin Using Poloxamer Gels for Local Bone Regeneration in a Rat Calvarial Defect Model.

Lactoferrin (LF) is a multifunctional milk glycoprotein that promotes bone regeneration. Local delivery of LF at the bone defect site is a promising approach for enhancement of bone regeneration, but efficient systems for sustained local delivery are still largely missing. The aim of this study was to investigate the potential of the poloxamers for sustained delivery of LF to enhance local bone regeneration. The developed LF/poloxamer formulations were liquid at room temperature (20 °C) transforming to a sustained releasing gel depot at body temperature (37 °C). In vitro release studies demonstrated an initial burst release (~50%), followed by slower release of LF for up to 72 h. Poloxamer, with and without LF, increased osteoblast viability at 72 h (p < 0.05) compared to control, and the immune response from THP-1 cells was mild when compared to the suture material. In rat calvarial defects, the LF/poloxamer group had lower bone volume than the controls (p = 0.0435). No difference was observed in tissue mineral density and lower bone defect coverage scores (p = 0.0267) at 12 weeks after surgery. In conclusion, LF/poloxamer formulations support cell viability and do not induce an unfavourable immune response; however, LF delivery via the current formulation of LF200/poloxamer gel did not demonstrate enhanced bone regeneration and was not compatible with the rat calvarial defect model.

Overlay repair with a synthetic collagen scaffold improves the quality of healing in a rat rotator cuff repair model.

BACKGROUND: Augmenting repairs with extracellular matrix-based scaffolds is a common option for rotator cuff tears. In this study, a new collagen scaffold was assessed for its efficacy in augmenting rotator cuff repair. METHODS: The collagen scaffold was assessed in vitro for cytocompatibility and retention of tenocyte phenotype using alamarBlue assays, fluorescent imaging, and real-time polymerase chain reaction. Immunogenicity was assessed in vitro by the activation of human monocytes. In vivo, by use of a modified rat rotator cuff defect model, supraspinatus tendon repairs were carried out in 40 animals. Overlay augmentation with the collagen scaffold was compared with unaugmented repairs. At 6 and 12 weeks postoperatively, the repairs were tested biomechanically to evaluate repair strength, as well as histologically to assess quality of healing. RESULTS: The collagen scaffold supported human tendon-derived cell growth in vitro, with cells demonstrating proliferation and appearing morphologically tenocytic over the experimental period. No immunogenic responses were provoked compared with suture material control. In vivo, augmentation with the scaffold improved the histologic scores at 12 weeks (8.4 of 15 vs 6.4 of 15, P = .032). However, no significant difference was detected with mechanical testing. CONCLUSION: The new collagen scaffold was supportive of cell growth in vitro and generated a minimal acute inflammatory response. In vivo, we observed an improvement in the histologic appearance of the repair at 12 weeks. However, a meaningful increase in biomechanical strength was not achieved. Further modification and improvement of the scaffold are required prior to consideration for clinical use.

Bovine bone particulates containing bone anabolic factors as a potential xenogenic bone graft substitute.

BACKGROUND: Alternative grafts are needed to improve the healing of bone non-union. Here, we assessed a bovine bone product which retains the inorganic and organic components of bone, as an alternative bone graft. METHODS: Bovine bone matrix proteins (BBMPs) were isolated from bovine bone particulates (BBPs) and tested in vitro. Primary rat osteoblast viability, differentiation, and mineralisation were assessed with alamarBlue®, real-time PCR, and von Kossa staining assays, respectively. Osteoclast formation was assessed in primary murine bone marrow cultures with TRAP staining. Human osteoblast growth and differentiation in the presence of BBPs was evaluated in 3D collagen gels in vitro using alamarBlue® and real-time PCR, respectively. The efficacy of BBPs as an alternative bone graft was tested in a rat critical-size calvarial defect model, with histology scored at 4 and 12 weeks post-surgery. RESULTS: In vitro, the highest concentration of BBMPs increased mineral deposition five-fold compared to the untreated control group (P < 0.05); enhanced the expression of key osteoblast genes encoding for RUNX2, alkaline phosphatase, and osteocalcin (P < 0.05); and decreased osteoclast formation three-fold, compared to the untreated control group (P < 0.05). However, the BBPs had no effect on primary human osteoblasts in vitro, and in vivo, no difference was found in healing between the BBP-treated group and the untreated control group. CONCLUSIONS: Overall, despite the positive effects of the BBMPs on the cells of the bone, the bovine bone product as a whole did not enhance bone healing. Finding a way to harness the positive effect of these BBMPs would provide a clear benefit for healing bone non-union.

Local application of lactoferrin promotes bone regeneration in a rat critical-sized calvarial defect model as demonstrated by micro-CT and histological analysis.

Lactoferrin is a multifunctional glycoprotein with therapeutic potential for bone tissue engineering. The aim of this study was to assess the efficacy of local application of lactoferrin on bone regeneration. Five-millimetre critical-sized defects were created over the right parietal bone in 64 Sprague-Dawley rats. The rats were randomized into four groups: group 1 (n  = â€…20) had empty defects; group 2 (n  = â€…20) had defects grafted with collagen gels (3 mg/ml); group 3 (n  = â€…20) had defects grafted with collagen gels impregnated with bovine lactoferrin (10 µg/gel); and group 4 (n  = â€…4) had sham surgeries (skin and periosteal incisions only). The rats were sacrificed at 4 or 12 weeks post-operatively, and the calvaria were excised and evaluated with micro-CT (Skyscan 1172) followed by histology. The bone volume fraction (BV/TV) was higher in lactoferrin-treated animals at both timepoints, with groups 1, 2, 3 and 4 measuring 10.5  ± â€…1.1%, 8.6  ± â€…1.4%, 16.5  ± â€…0.6% and 24.27  ± â€…2.6%, respectively, at 4 weeks (P  < â€…0.05); and 12.2  ± â€…1.3%, 13.6  ± â€…1.5%, 21.9  ± â€…1.2% and 29.3  ± â€…0.8%, respectively, at 12 weeks (P  < â€…0.05). Histological analysis revealed that the newly formed bone within the calvarial defects of all groups was a mixture of woven and lamellar bone, with more bone in the group treated with lactoferrin at both timepoints. Our study demonstrated that local application of lactoferrin significantly increased bone regeneration in a rat critical-sized calvarial defect model. The profound effect of lactoferrin on bone regeneration has therapeutic potential to improve the poor clinical outcomes associated with bony non-union. LF In Vivo JTERM Authors Contributions. Copyright © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Reduced Bone Density and Cortical Bone Indices in Female Adiponectin-Knockout Mice.

A positive association between fat and bone mass is maintained through a network of signaling molecules. Clinical studies found that the circulating levels of adiponectin, a peptide secreted from adipocytes, are inversely related to visceral fat mass and bone mineral density, and it has been suggested that adiponectin contributes to the coupling between fat and bone. Our study tested the hypothesis that adiponectin affects bone tissue by comparing the bone phenotype of wild-type and adiponectin-knockout (APN-KO) female mice between the ages of 8-37 weeks. Using a longitudinal study design, we determined body composition and bone density using dual energy x-ray absorptiometry. In parallel, groups of animals were killed at different ages and bone properties were analyzed by microcomputed tomography, dynamic histomorphometry, 3-point bending test, nanoindentation, and computational modelling. APN-KO mice had reduced body fat and decreased whole-skeleton bone mineral density. Microcomputed tomography analysis identified reduced cortical area fraction and average cortical thickness in APN-KO mice in all the age groups and reduced trabecular bone volume fraction only in young APN-KO mice. There were no major differences in bone strength and material properties between the 2 groups. Taken together, our results demonstrate a positive effect of adiponectin on bone geometry and density in our mouse model. Assuming adiponectin has similar effects in humans, the low circulating levels of adiponectin associated with increased fat mass are unlikely to contribute to the parallel increase in bone mass. Therefore, adiponectin does not appear to play a role in the coupling between fat and bone tissue.

Augmentation with an ovine forestomach matrix scaffold improves histological outcomes of rotator cuff repair in a rat model.

BACKGROUND: Rotator cuff tears can cause significant pain and functional impairment. Without surgical repair, the rotator cuff has little healing potential, and following surgical repair, they are highly prone to re-rupture. Augmenting such repairs with a biomaterial scaffold has been suggested as a potential solution. Extracellular matrix (ECM)-based scaffolds are the most commonly used rotator cuff augments, although to date, reports on their success are variable. Here, we utilize pre-clinical in vitro and in vivo assays to assess the efficacy of a novel biomaterial scaffold, ovine forestomach extracellular matrix (OFM), in augmenting rotator cuff repair. METHODS: OFM was assessed in vitro for primary tenocyte growth and adherence, and for immunogenicity using an assay of primary human dendritic cell activation. In vivo, using a murine model, supraspinatus tendon repairs were carried out in 34 animals. Augmentation with OFM was compared to sham surgery and unaugmented control. At 6- and 12-week time points, the repairs were analysed biomechanically for strength of repair and histologically for quality of healing. RESULTS: OFM supported tenocyte growth in vitro and did not cause an immunogenic response. Augmentation with OFM improved the quality of healing of the repaired tendon, with no evidence of excessive inflammatory response. However, there was no biomechanical advantage of augmentation. CONCLUSIONS: The ideal rotator cuff tendon augment has not yet been identified or clinically implemented. ECM scaffolds offer a promising solution to a difficult clinical problem. Here, we have shown improved histological healing with OFM augmentation. Identifying materials that offset the poorer mechanical properties of the rotator cuff post-injury/repair and enhance organised tendon healing will be paramount to incorporating augmentation into surgical treatment of the rotator cuff.