Scaffold-guided bone regeneration in large volume tibial segmental defects.

Large volume losses in weight bearing long bones are a major challenge in clinical practice. Despite multiple innovations over the last decades, significant limitations subsist in current clinical treatment options which is driving a strong clinical demand for clinically translatable treatment alternatives, including bone tissue engineering applications. Despite these shortcomings, preclinical large animal models of large volume segmental bone defects to investigate the regenerative capacity of bone tissue engineering strategies under clinically relevant conditions are rarely described in literature. We herein present a newly established preclinical ovine animal model for the treatment of XL volume (19 cm3) segmental tibial defects. In eight aged male Merino sheep (age > 6 years) a mid-diaphyseal tibial segmental defect was created and stabilized with a 5.6 mm Dynamic Compression Plate (DCP). We present short-term (3 months) and long-term (12-15 months) results of a pilot study using medical grade Polycaprolactone-Tricalciumphosphate (mPCL-TCP) scaffolds combined with a dose of 2 mg rhBMP-7 delivered in Platelet-Rich- Plasma (PRP). Furthermore, detailed analyses of the mechanical properties of the scaffolds as well as interfragmentary movement (IFM) and DCP-surface strain in vitro and a comprehensive description of the surgical and post-surgery protocol and post-mortem analysis is given.

A preclinical large-animal model for the assessment of critical-size load-bearing bone defect reconstruction.

Critical-size bone defects, which require large-volume tissue reconstruction, remain a clinical challenge. Bone engineering has the potential to provide new treatment concepts, yet clinical translation requires anatomically and physiologically relevant preclinical models. The ovine critical-size long-bone defect model has been validated in numerous studies as a preclinical tool for evaluating both conventional and novel bone-engineering concepts. With sufficient training and experience in large-animal studies, it is a technically feasible procedure with a high level of reproducibility when appropriate preoperative and postoperative management protocols are followed. The model can be established by following a procedure that includes the following stages: (i) preoperative planning and preparation, (ii) the surgical approach, (iii) postoperative management, and (iv) postmortem analysis. Using this model, full results for peer-reviewed publication can be attained within 2 years. In this protocol, we comprehensively describe how to establish proficiency using the preclinical model for the evaluation of a range of bone defect reconstruction options.

Efficacy of novel synthetic bone substitutes in the reconstruction of large segmental bone defects in sheep tibiae.

The treatment of large bone defects, particularly those with segmental bone loss, remains a significant clinical challenge as current approaches involving surgery or bone grafting often do not yield satisfactory long-term outcomes. This study reports the evaluation of novel ceramic scaffolds applied as bone graft substitutes in a clinically relevant in vivo model. Baghdadite scaffolds, unmodified or modified with a polycaprolactone coating containing bioactive glass nanoparticles, were implanted into critical-sized segmental bone defects in sheep tibiae for 26 weeks. Radiographic, biomechanical, µ-CT and histological analyses showed that both unmodified and modified baghdadite scaffolds were able to withstand physiological loads at the defect site, and induced substantial bone formation in the absence of supplementation with cells or growth factors. Notably, all samples showed significant bridging of the critical-sized defect (average 80%) with evidence of bone infiltration and remodelling within the scaffold implant. The unmodified and modified baghdadite scaffolds achieved similar outcomes of defect repair, although the latter may have an initial mechanical advantage due to the nanocomposite coating. The baghdadite scaffolds evaluated in this study hold potential for use as purely synthetic bone graft substitutes in the treatment of large bone defects while circumventing the drawbacks of autografts and allografts.

The metaphyseal bone defect in distal radius fractures and its implication on trabecular remodeling-a histomorphometric study (case series).

BACKGROUND: The invention of the locking plate technology leads to alterations of treatment strategies at metaphyseal fracture sites with the concept of spontaneous remodeling of trabecular bone voids. Whereas trabecular regeneration has been proven in experimental animal studies, no histologic data exist on human fracture healing with special emphasis on bone voids. METHODS: In order to qualify the trabecular bone remodeling capacity in vivo, bone specimens from the metaphyseal bone void were analyzed 14 months after trauma using quantitative histomorphometry. Twenty-five patients with an unstable dorsally displaced distal radius fracture were fixed with a palmar locking plate without additional bone graft or substitute. At implant removal, specimens from the previous compression void were harvested with a trephine in a volar-dorsal direction. In 16 patients, histomorphometric analysis could be performed, comparing the dorsal trabecular network with the volar, non-compressed ultrastructure. RESULTS: Significant differences for bone volume/total volume (BV/TV), trabecular number (TbN) and trabecular separation (TbSp), but not for trabecular thickness (TbTh) and osteoid volume/total volume (OV/TV), were detected. Neither patient age, defect size nor gender had a significant influence on bone remodeling. CONCLUSIONS: The results of this study indicate that trabecular bone remodeling does not lead to pre-trauma bone quality in metaphyseal bone compression voids following reduction and application of a locking plate.

Delayed minimally invasive injection of allogenic bone marrow stromal cell sheets regenerates large bone defects in an ovine preclinical animal model.

Cell-based tissue engineering approaches are promising strategies in the field of regenerative medicine. However, the mode of cell delivery is still a concern and needs to be significantly improved. Scaffolds and/or matrices loaded with cells are often transplanted into a bone defect immediately after the defect has been created. At this point, the nutrient and oxygen supply is low and the inflammatory cascade is incited, thus creating a highly unfavorable microenvironment for transplanted cells to survive and participate in the regeneration process. We therefore developed a unique treatment concept using the delayed injection of allogenic bone marrow stromal cell (BMSC) sheets to regenerate a critical-sized tibial defect in sheep to study the effect of the cells' regeneration potential when introduced at a postinflammatory stage. Minimally invasive percutaneous injection of allogenic BMSCs into biodegradable composite scaffolds 4 weeks after the defect surgery led to significantly improved bone regeneration compared with preseeded scaffold/cell constructs and scaffold-only groups. Biomechanical testing and microcomputed tomography showed comparable results to the clinical reference standard (i.e., an autologous bone graft). To our knowledge, we are the first to show in a validated preclinical large animal model that delayed allogenic cell transplantation can provide applicable clinical treatment alternatives for challenging bone defects in the future.

Estrogen Deficiency-Associated Bone Loss in the Maxilla: A Methodology to Quantify the Changes in the Maxillary Intra-radicular Alveolar Bone in an Ovariectomized Rat Osteoporosis Model.

The effects of estrogen deficiency on bone characteristics are site-dependent, with the most commonly studied sites being appendicular long bones (proximal femur and tibia) and axial bones (vertebra). The effect on the maxillary and mandibular bones is still inconsistent and requires further investigation. This study was designed to evaluate bone quality in the posterior maxilla of ovariectomized rats to validate this site as an appropriate model to study the effect of osteoporotic changes. Forty-eight 3-month-old female Sprague-Dawley rats were randomly divided into two groups: an ovariectomized (OVX) group (n=24) and Sham-operated (SHAM) group (n=24). Six rats were randomly sacrificed from both groups at time points 8, 12, 16, and 20 weeks. The samples from tibia and maxilla were collected for micro computed tomography (µCT) and histological analysis. For the maxilla, the volume of interest area focused on the furcation areas of the first and second molar. Trabecular bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and connectivity density (Conn.Dens) were analyzed after Micro CT scanning. At 8 weeks the indices BV/TV, Tb.Sp., Tb.N., and Conn.Dens showed significant differences (p<0.05) between the OVX and SHAM groups in the tibia. Compared with the tibia, the maxilla developed osteoporosis at a later stage, with significant changes in maxillary bone density only occurring after 12 weeks. Compared with the SHAM group, both the first and second molars of the OVX group showed significantly decreased BV/TV values from 12 weeks, and these changes were sustained through 16 and 20 weeks. For Tb.Sp., there were significant increases in bone values for the OVX group compared with the SHAM group at 12, 16, and 20 weeks. Histological changes were highly consistent with Micro CT results. This study established a method to quantify the changes of intra-radicular alveolar bone in the posterior maxilla in an accepted rat osteoporosis model. The degree of the osteoporotic changes to trabecular bone architecture is site-dependent and at least 3 months are required for the osteoporotic effects to be apparent in the posterior maxilla following rat OVX.

Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification.

The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80(+)Mac-2(+)) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80(+)Mac-2(neg)), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window.

Bone Regeneration Based on Tissue Engineering Conceptions - A 21st Century Perspective.

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental "origin" require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

ß-glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice.

OBJECTIVE: The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 ß-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-ß-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS: SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS: After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION: Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic ß-glucan or mannan exposure.

Distribution of bone marrow-derived cells in the fracture callus during plate fixation in a green fluorescent protein-chimeric mouse model.

To clarify the distribution of bone-marrow-derived cells in fractures treated by plate fixation, fracture models were created using the green fluorescent protein (GFP) chimeric mouse. We observed 2 types of fracture healing processes with different types of callus formation and cellular events by using Mouse Fix™, a device allowing plate fixation on the mouse femur, and differences in the distribution of bone-marrow-derived cells between the 2 types. The GFP chimeric mice were created by bone marrow transplantation. Fractures were created on the left femurs of mice and stabilized with either rigid (Group R) or flexible (Group F) plates to prepare undecalcified fresh-frozen sections. In Group F, a large external callus and a large intramedullary callus were formed mostly by endochondral ossification. The cells that made up the intramedullary callus and callus in the fracture gap were GFP positive, but most cells of the external callus were not. In Group R, bone union was achieved mostly without external callus formation, bone apposition occurred directly in the gap, and a small intramedullary callus was formed. As observed in Group F, this group had GFP-positive cells in the callus within the fracture gap and in the intramedullary calluses. The results of this study provided direct evidence of the distribution of bone-marrow-derived cells in the callus of fractures treated by plate fixation under different stability conditions.