In-situ tissue regeneration through SDF-1α driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect.

In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required. STATEMENT OF SIGNIFICANCE: Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.

Intraosseous Delivery of Bone Morphogenic Protein-2 Using a Self-Assembling Peptide Hydrogel.

Osteonecrosis of the femoral head (ONFH) is a debilitating hip disorder, which often produces a permanent femoral head deformity and osteoarthritis. The local delivery of biological agents capable of stimulating bone healing offer potential new treatment options for patients with ONFH. Previous studies from our laboratory have shown that a local intraosseous infusion of bone morphogenic protein-2 (BMP-2) was effective in stimulating new bone formation in a piglet model of ischemic ONFH. However, infusion of BMP-2 solution was associated with unwanted dissemination of BMP-2 out of the femoral head and produced heterotopic ossification in the hip capsule. Injectable hydrogels offer a potential method to control the dissemination of biological molecules in vivo. In the present study, we evaluated the potential of a peptide-based, self-assembling hydrogel called RADA16 to transition from a solution to a gel following infusion into the femoral head, thereby preventing backflow, as well as its potential use as a delivery vehicle for BMP-2. Cadaver pig femoral heads were used to study the backflow and the distribution of RADA16 following an intraosseous infusion. Microcomputed tomography analysis following the infusion of RADA16 mixed with a radiocontrast agent revealed a significant decrease in the amount of back flow of radiocontrast agent down the needle track compared to the soluble infusion of radiocontrast without RADA16. Furthermore, RADA16 mixed with radiocontrast agent showed good distribution within the femoral head. In addition, in vitro experiments revealed that higher concentrations of RADA16 decreased the rate of BMP-2 dissemination out of the hydrogel. The BMP-2 that was released from RADA16 maintains its biological activity, inducing the phosphorylation of SMAD1/5/8 in pig primary bone marrow stromal cells. Lastly, pig primary bone marrow stromal cells showed significantly increased in vitro proliferation on RADA16 hydrogels over 1 week compared to tissue culture plastic, suggesting that it is a suitable matrix for supporting cellular proliferation. In conclusion, RADA16 showed potential for use as a drug delivery vehicle to control the delivery of BMP-2 within the femoral head. This novel therapy may be able to provide benefits to patients suffering from debilitating conditions such as osteonecrosis of the femoral head.

Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation.

The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

Clotting Mimicry from Robust Hemostatic Bandages Based on Self-Assembling Peptides.

Uncontrolled bleeding from traumatic wounds is a major factor in deaths resulting from military conflict, accidents, disasters and crime. Self-assembling peptide nanofibers have shown superior hemostatic activity, and herein, we elucidate their mechanism by visualizing the formation of nanofiber-based clots that aggregate blood components with a similar morphology to fibrin-based clots. Furthermore, to enhance its direct application to a wound, we developed layer-by-layer assembled thin film coatings onto common materials used for wound dressings-gauze and gelatin sponges. We find these nanofibers elute upon hydration under physiological conditions and generate nanofiber-based clots with blood. After exposure to a range of harsh temperature conditions (-80 to 60 °C) for a week and even 5 months at 60 °C, these hemostatic bandages remain capable of releasing active nanofibers. In addition, the application of these nanofiber-based films from gauze bandages was found to accelerate hemostasis in porcine skin wounds as compared to plain gauze. The thermal robustness, in combination with the self-assembling peptide's potent hemostatic activity, biocompatibility, biodegradability, and low cost of production, makes this a promising approach for a cheap yet effective hemostatic bandage.

The collagen I mimetic peptide DGEA enhances an osteogenic phenotype in mesenchymal stem cells when presented from cell-encapsulating hydrogels.

Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.

Local delivery of VEGF and SDF enhances endothelial progenitor cell recruitment and resultant recovery from ischemia.

Biomaterials may improve outcomes of endothelial progenitor-based therapies for the treatment of ischemic cardiovascular disease, due to their ability to direct cell behavior. We hypothesized that local, sustained delivery of exogenous vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF) from alginate hydrogels could increase recruitment of systemically infused endothelial progenitors to ischemic tissue, and subsequent neovascularization. VEGF and SDF were found to enhance in vitro adhesion and migration of outgrowth endothelial cells (OECs) and circulating angiogenic cells (CACs), two populations of endothelial progenitors, by twofold to sixfold, and nearly doubled recruitment to both ischemic and nonischemic muscle tissue in vivo. Local delivery of VEGF and SDF to ischemic hind-limbs in combination with systemic CAC delivery significantly improved functional perfusion recovery over OEC delivery, or either treatment alone. Compared with OECs, CACs were more responsive to VEGF and SDF treatment, promoted in vitro endothelial sprout formation in a paracrine manner more potently, and demonstrated greater influence on infiltrating inflammatory cells in vivo. These studies demonstrate that accumulation of infused endothelial progenitors can be enriched using biomaterial-based delivery of VEGF and SDF, and emphasize the therapeutic benefit of using CACs for the treatment of ischemia.

Biphasic ferrogels for triggered drug and cell delivery.

Ferrogels are an attractive material for many biomedical applications due to their ability to deliver a wide variety of therapeutic drugs on-demand. However, typical ferrogels have yet to be optimized for use in cell-based therapies, as they possess limited ability to harbor and release viable cells. Previously, an active porous scaffold that exhibits large deformations and enhanced biological agent release under moderate magnetic fields has been demonstrated. Unfortunately, at small device sizes optimal for implantation (e.g., 2 mm thickness), these monophasic ferrogels no longer achieve significant deformation due to a reduced body force. A new biphasic ferrogel, containing an iron oxide gradient, capable of large deformations and triggered release even at small gel dimensions, is presented in this study. Biphasic ferrogels demonstrate increased porosity, enhanced mechanical properties, and potentially increased biocompatibility due to their reduced iron oxide content. With their ability to deliver drugs and cells on-demand, it is expected that these ferrogels will have wide utility in the fields of tissue engineering and regenerative medicine.

Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats.

OBJECTIVES: Calcium phosphate cement (CPC) is promising for dental and craniofacial applications due to its ability to be injected or filled into complex-shaped bone defects and molded for esthetics, and its resorbability and replacement by new bone. The objective of this study was to investigate bone regeneration via novel macroporous CPC containing absorbable fibers, hydrogel microbeads and growth factors in critical-sized cranial defects in rats. METHODS: Mannitol porogen and alginate hydrogel microbeads were incorporated into CPC. Absorbable fibers were used to provide mechanical reinforcement to CPC scaffolds. Six CPC groups were tested in rats: (1) control CPC without macropores and microbeads; (2) macroporous CPC+large fiber; (3) macroporous CPC+large fiber+nanofiber; (4) same as (3), but with rhBMP2 in CPC matrix; (5) same as (3), but with rhBMP2 in CPC matrix+rhTGF-ß1 in microbeads; (6) same as (3), but with rhBMP2 in CPC matrix+VEGF in microbeads. Rats were sacrificed at 4 and 24 weeks for histological and micro-CT analyses. RESULTS: The macroporous CPC scaffolds containing porogen, absorbable fibers and hydrogel microbeads had mechanical properties similar to cancellous bone. At 4 weeks, the new bone area fraction (mean±sd; n=5) in CPC control group was the lowest at (14.8±3.3)%, and that of group 6 (rhBMP2+VEGF) was (31.0±13.8)% (p<0.05). At 24 weeks, group 4 (rhBMP2) had the most new bone of (38.8±15.6)%, higher than (12.7±5.3)% of CPC control (p<0.05). Micro-CT revealed nearly complete bridging of the critical-sized defects with new bone for several macroporous CPC groups, compared to much less new bone formation for CPC control. SIGNIFICANCE: Macroporous CPC scaffolds containing porogen, fibers and microbeads with growth factors were investigated in rat cranial defects for the first time. Macroporous CPCs had new bone up to 2-fold that of traditional CPC control at 4 weeks, and 3-fold that of traditional CPC at 24 weeks, and hence may be useful for dental, craniofacial and orthopedic applications.

Presentation of BMP-2 mimicking peptides in 3D hydrogels directs cell fate commitment in osteoblasts and mesenchymal stem cells.

Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.

Rapid and extensive collapse from electrically responsive macroporous hydrogels.

Electrically responsive hydrogels are created with interconnected macropores, which greatly enhance their ability to rapidly undergo volumetric collapse when subjected to moderate electric fields. When optimized, these electrogels are easily integrated into arrays capable of rapid configurational and chromatic optical modulations, and when loaded with drugs, are able to coordinate the delivery profile of multiple drugs.

Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae.

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with ß-tricalcium phosphate microparticles (mPCL-TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL-TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.

Performance and biocompatibility of extremely tough alginate/polyacrylamide hydrogels.

Although hydrogels now see widespread use in a host of applications, low fracture toughness and brittleness have limited their more broad use. As a recently described interpenetrating network (IPN) of alginate and polyacrylamide demonstrated a fracture toughness of ≈ 9000 J/m(2), we sought to explore the biocompatibility and maintenance of mechanical properties of these hydrogels in cell culture and in vivo conditions. These hydrogels can sustain a compressive strain of over 90% with minimal loss of Young's Modulus as well as minimal swelling for up to 50 days of soaking in culture conditions. Mouse mesenchymal stem cells exposed to the IPN gel-conditioned media maintain high viability, and although cells exposed to conditioned media demonstrate slight reductions in proliferation and metabolic activity (WST assay), these effects are abrogated in a dose-dependent manner. Implantation of these IPN hydrogels into subcutaneous tissue of rats for 8 weeks led to mild fibrotic encapsulation and minimal inflammatory response. These results suggest the further exploration of extremely tough alginate/PAAM IPN hydrogels as biomaterials.

In vivo tracking of segmental bone defect healing reveals that callus patterning is related to early mechanical stimuli.

This study addresses the hypothesis that callus formation, patterning, and mineralisation are impaired during the early phase of critical sized bone defect healing, and may relate to inter-fragmentary tissue strains within the bone defect area. Twenty four 12 week old Sprague Dawley rats were used for this study. They were divided into two groups defined by the femur bone defect size: (i) 1 mm resulting in normal healing (NH), and (ii) a large sized 5 mm defect resulting in critical healing (CH). Callus formation, patterning, and mineralisation kinetics in both groups were examined in the periosteal and osteotomy gap regions using a novel longitudinal study setup. Finite element analyses on µCT generated tomograms were used to determine inter-fragmentary tissue strain patterns and compared to callus formation and patterning over the course of time. Using a novel longitudinal study technique with µCT, in vivo tracking and computer simulation approaches, this study demonstrates that: (i) periosteal bone formation and patterning are significantly influenced by bone defect size as early as 2 weeks; (ii) osteotomy gap callus formation and patterning are influenced by bone defect size, and adapt towards a non-union in critical cases by deviating into a medullary formation route as early as 2 weeks after osteotomy; (iii) the new bone formation in the osteotomy gap enclosing the medullary cavity in the CH group is highly mineralised; (iv) inter-fragmentary strain patterns predicted during the very early soft callus tissue phase (less than 2 weeks) are concurrent with callus formation and patterning at later stages. In conclusion, bone defect size influences early onset of critical healing patterns.

A tissue engineering solution for segmental defect regeneration in load-bearing long bones.

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow-derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep--a model closely resembling human bone formation and structure--were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Intra-operatively customized implant coating strategies for local and controlled drug delivery to bone.

Bone is one of the few tissues in the human body with high endogenous healing capacity. However, failure of the healing process presents a significant clinical challenge; it is a tremendous burden for the individual and has related health and economic consequences. To overcome such healing deficits, various concepts for a local drug delivery to bone have been developed during the last decades. However, in many cases these concepts do not meet the specific requirements of either surgeons who must use these strategies or individual patients who might benefit from them. We describe currently available methods for local drug delivery and their limitations in therapy. Various solutions for drug delivery to bone focusing on clinical applications and intra-operative constraints are discussed and drug delivery by implant coating is highlighted. Finally, a new set of design and performance requirements for intra-operatively customized implant coatings for controlled drug delivery is proposed. In the future, these requirements may improve approaches for local and intra-operative treatment of patients.

Biomaterial delivery of morphogens to mimic the natural healing cascade in bone.

Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing.

Influence of prosthesis design and implantation technique on implant stresses after cementless revision THR.

BACKGROUND: Femoral offset influences the forces at the hip and the implant stresses after revision THR. For extended bone defects, these forces may cause considerable bending moments within the implant, possibly leading to implant failure. This study investigates the influences of femoral anteversion and offset on stresses in the Wagner SL revision stem implant under varying extents of bone defect conditions. METHODS: Wagner SL revision stems with standard (34 mm) and increased offset (44 mm) were virtually implanted in a model femur with bone defects of variable extent (Paprosky I to IIIb). Variations in surgical technique were simulated by implanting the stems each at 4° or 14° of anteversion. Muscle and joint contact forces were applied to the reconstruction and implant stresses were determined using finite element analyses. RESULTS: Whilst increasing the implant's offset by 10 mm led to increased implant stresses (16.7% in peak tensile stresses), altering anteversion played a lesser role (5%). Generally, larger stresses were observed with reduced bone support: implant stresses increased by as much as 59% for a type IIIb defect. With increased offset, the maximum tensile stress was 225 MPa. CONCLUSION: Although increased stresses were observed within the stem with larger offset and increased anteversion, these findings indicate that restoration of offset, key to restoring joint function, is unlikely to result in excessive implant stresses under routine activities if appropriate fixation can be achieved.

Influence of gender and fixation stability on bone defect healing in middle-aged rats: a pilot study.

BACKGROUND: Gender and stability of fixation independently influence bone regeneration but their combined effects are unclear. QUESTIONS/PURPOSES: In a pilot study we determined the combined influence of gender and fixation stability on the callus of middle-aged rats regarding (1) biomechanical properties; (2) bridging over time; (3) callus formation; and (4) callus size, geometry, mineralization, and microstructure. METHODS: We osteotomized the left femur of 32 Sprague-Dawley rats (12 months old). Femurs were externally fixed with a gap of 1.5 mm in four groups of eight animals each: female semirigid, male semirigid, female rigid, and male rigid. Qualitative and quantitative in vivo radiographic analyses were performed twice weekly. Six weeks postoperatively, harvested femora were evaluated using micro-CT and biomechanical testing. RESULTS: Torsional stiffness and maximum torque at failure were higher in male and in semirigidly fixed fractures. Radiographic analysis revealed earlier bridging and callus formation in both male groups. Micro-CT analysis showed a larger callus size, altered geometry, and microstructure in males and semirigidly fixed animals, whereas mineralization was similar in all animals. CONCLUSION: Our data suggest female gender represents an independent risk factor for bone healing in middle-aged rats. Although healing in females was delayed compared with males, they exhibited a similar response (superior callus properties) to a more semirigid fixation. CLINICAL RELEVANCE: While female gender appears to reflect a risk for impaired bone healing in middle-aged female rats, clinical studies would be required to confirm the finding in humans.

Fracture healing is accelerated in the absence of the adaptive immune system.

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild-type (WT) and recombination activating gene 1 knockout (RAG1(-/-)) mice lacking the adaptive immune system. Healing was investigated using micro-computed tomography (µCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1(-/-) mice compared to the WT group. µCT evaluation of RAG1(-/-) specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1(-/-) compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1(-/-) mice than in the WT mice. Endochondral ossification was accelerated in the RAG1(-/-) mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1(-/-) mice. In contrast, expression of anti-inflammatory interleukin 10 (IL-10) was strongly upregulated in RAG1(-/-) mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1(-/-) mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti-inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair.

A 5-mm femoral defect in female but not in male rats leads to a reproducible atrophic non-union.

INTRODUCTION: The objectives of this study were to (1) establish a reproducible atrophic non-union model in rats by creation of a segmental femoral bone defect that allows, (2) in-depth characterization of impaired healing, and (3) contrast its healing patterns to the normal course. Hypothesis was that a 5-mm bone defect in male rats would deviate from uneventful healing patterns and result in an atrophic non-union. MATERIALS AND METHODS: A femoral osteotomy was performed in two groups of 12-week-old male rats (1 vs. 5 mm gap) stabilized with an external fixator. Bone healing in these models was evaluated by radiology, biomechanics, and histology at 6 or 8 weeks. The evaluation of the 5-mm group revealed in some cases a delayed rather than a non-union, and therefore, a group of female counterparts was included. RESULTS: The creation of a 5-mm defect in female rats resulted in a reproducible atrophic non-union characterized by sealing of the medullary canal, lack of cartilage formation, and negligible mechanical properties of the callus. In both gap size models, the male subjects showed advanced healing compared to females. DISCUSSION AND CONCLUSION: This study showed that even under uneventful healing conditions in terms of age and bone defect size, there is a sex-specific advanced healing in male compared to female subjects. Contrary to our initial hypothesis, only the creation of a 5-mm segmental femoral defect in female rats led to a reproducible atrophic non-union. It has been shown that an atrophic non-union exhibits different healing patterns compared to uneventful healing. A total lack of endochondral bone formation, soft tissue prolapse into the defect, and bony closure of the medullary cavity have been shown to occur in the non-union model.