Treatment Of Large Bone Defects Research Articles

Rat Calvarial Bone Regeneration by 3D-Printed Beta-Tricalcium Phosphate Incorporating MicroRNA-200c

Advanced fabrication methods of bone grafts designed to match defect sites that combine biodegradable, osteoconductive materials with potent, osteoinductive biologics would significantly impact the clinical treatment of large bone defects. In this study, we engineered synthetic bone grafts using a hybrid material approach that combined 3D-printed biodegradable, osteoconductive β-tricalcium phosphate (β-TCP) with osteoinductive microRNA(miR)-200c. 3D-printed β-TCP scaffolds were fabricated utilizing suspension-enclosing projection-stereolithography (SEPS) process to produce constructs with reproducible microarchitectures that enhanced the osteoconductive properties of β-TCP. 3D-printed β-TCP scaffolds coated with miR-200c incorporated collagen increased the transfection efficiency of miR-200c and osteogenic differentiation of hBMSCs in vitro. Furthermore, these hybrid material, miR-incorporated scaffolds significantly enhanced bone regeneration in critical-sized rat calvarial defects. These results strongly indicate that through use of SEPS 3D-printing technology, hybrid scaffolds combined by osteoconductive biomaterials and osteoinductive biologics can be used as superior bone substitutes for the clinical treatment of large bone defects.

Impact of scaffold granule size use in Masquelet technique on periosteal reaction: a study in rat femur critical size bone defect model

PurposeThe Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. Compared to mature periosteum, the induced membrane differs significantly. However, both play a crucial role in bone regeneration. As part of a histological and radiological post-evaluation of an earlier project, we analyzed the influence of the granule size of the bone void filler Herafill® on development of periosteum regrowth in a critical size defect.MethodsWe compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) in vivo in a rat femoral critical size defect (10 mm) treated with the induced membrane technique. After 8 weeks healing time, femurs were harvested and taken for histological and radiological analysis.ResultsA significantly increased regrowth of periosteum into the defect was found when small granules were used. Large granules showed significantly increased occurrence of bone capping. Small granules lead to significant increase in callus formation in the vicinity to the membrane.ConclusionThe size of Herafill® granules has significant impact on the development of periosteal-like structures around the defect using Masquelet’s induced membrane technique. Small granules show significantly increased regrowth of periosteum and improved bone formation adjacent to the induced membrane.

3D Biomimetic Porous Titanium (Ti6Al4V ELI) Scaffolds for Large Bone Critical Defect Reconstruction: An Experimental Study in Sheep

Simple SummaryThe authors propose a new reconstructive technique that proved to be suitable to reach this purpose through the use of a custom-made biomimetic porous titanium scaffold. An in vivo study was undertaken where a complete critical defect was experimentally created in the diaphysis of the right tibia of twelve sheep and replaced with a five-centimeter porous scaffold of electron beam melting (EBM)-manufactured titanium alloy or a porous hydroxyapatite scaffold. Our results show that EBM-formed titanium devices, if used to repair critical bone defects in a large animal model, can guarantee immediate body weight-bearing, a rapid functional recovery, and a good osseointegration. The porous hydroxyapatite scaffolds proved to be not suitable in this model of large bone defect due to their known poor mechanical properties.The main goal in the treatment of large bone defects is to guarantee a rapid loading of the affected limb. In this paper, the authors proposed a new reconstructive technique that proved to be suitable to reach this purpose through the use of a custom-made biomimetic porous titanium scaffold. An in vivo study was undertaken where a complete critical defect was experimentally created in the diaphysis of the right tibia of twelve sheep and replaced with a five-centimeter porous scaffold of electron beam melting (EBM)-sintered titanium alloy (EBM group n = 6) or a porous hydroxyapatite scaffold (CONTROL group, n = 6). After surgery, the sheep were allowed to move freely in the barns. The outcome was monitored for up to 12 months by periodical X-ray and clinical examination. All animals in the CONTROL group were euthanized for humane reasons within the first month after surgery due to the onset of plate bending due to mechanical overload. Nine months after surgery, X-ray imaging showed the complete integration of the titanium implant in the tibia diaphysis and remodeling of the periosteal callus, with a well-defined cortical bone. At 12 months, sheep were euthanized, and the tibia were harvested and subjected to histological analysis. This showed bone tissue formations with bone trabeculae bridging titanium trabeculae, evidencing an optimal tissue-metal interaction. Our results show that EBM-sintered titanium devices, if used to repair critical bone defects in a large animal model, can guarantee immediate body weight-bearing, a rapid functional recovery, and a good osseointegration. The porous hydroxyapatite scaffolds proved to be not suitable in this model of large bone defect due to their known poor mechanical properties.

The effect of induced membranes combined with enhanced bone marrow and 3D PLA-HA on repairing long bone defects in vivo.

The repair of large bone defects has always been a challenge, especially with respect to regeneration capacity and autogenous bone availability. To address this problem, we fabricated a 3D-printed polylactic acid (PLA) and hydroxyapatite (HA) scaffold (3D-printed PLA-HA, providing scaffold) loaded with enhanced bone marrow (eBM, providing seed cells) combined with induced membrane (IM, providing grow factors) to repair large radial defects in rabbits. in vitro assays, we demonstrated that 3D-printed PLA-HA had excellent biocompatibility, as shown by co-culturing with mesenchymal stem cells (MSCs); eBM-derived MSCs exhibited considerable differentiation potential, as shown in trilineage differentiation assays. To investigate bone formation efficacy in vivo, the rabbit radial long bone defect model was established. In the first stage, polymethylmethacrylate (PMMA) was inserted into the bone defect to stimulate the formation of IM; in the second stage, iliac crest bone graft (ICBG) with IM, PLA-HA alone with the removal of IM, PLA-HA with IM, and PLA-HA in conjunction with IM and eBM were sequentially applied to repair the long bone defect. At 8, 12, and 16 weeks, X-ray plain radiography, microcomputed tomography, and histological analysis were performed to evaluate the efficacy of bone repair and bone regeneration in each group. We found that IM combined with PLA-HA and eBM prominently enhanced bone repair and reconstruction, equivalent to that of IM/ICBG. Taken together, the data suggest that PLA-HA loaded with eBM combined with IM can be an alternative to IM with bone autografts for the treatment of large bone defects.

Investigation of bone reconstruction using an attenuated immunogenicity xenogenic composite scaffold fabricated by 3D printing

Bone is known to have a natural function to heal itself. However, if the bone damage is beyond a critical degree, intervention such as bone grafting may be imperative. In this work, the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone (PCL) using 3D printing is put forward. α1, 3-galactosyltransferase deficient pigs were used as the donor source of a xenograft. Decellularized porcine bone (DCB) with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone regeneration. The 3D printed DCB-PCL scaffolds combined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive strength. The biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion, viability, alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem cells. The in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation, which is correlated to the DCB content. Furthermore, critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in vivo. The results confirm that in comparison with PCL scaffolds, the DCB-PCL scaffolds can significantly improve new bone formation in cranial defects. Thus, the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration alternative in the clinical treatment of large bone defects.

An Analysis of Complications and Bone Defect Length With the Use of Induced Membrane Technique in the Upper Limb: A Systematic Review.

Background: The induced membrane technique was originally described as a technique for the reconstruction of long bone defects. The authors performed a systematic review to determine whether the use of the induced membrane technique is effective in large bony defects in the upper extremity. Methods: A qualitative systematic review was conducted using PubMed, EBSCO, and Google Scholar databases to record all studies reporting on complications of the induced membrane technique in the upper extremity. Studies written after 1990 in English language journals met the inclusion criteria. Exclusion criteria were non-English language publications, animal studies, failure to identify the location of the bone defect, failure to identify whether complications were associated with the procedure, and failure to define the length of bone defect. Results: A total of 1422 studies were identified in the original search. Twelve studies satisfied the criteria for inclusion. A total of 70 patients with 83 upper extremity cases were reported: 1 proximal interphalangeal joint, 22 phalanges, 8 metacarpals, 37 forearms, 14 humeri, and 1 clavicle. The mean bone defect size was 4.0 cm (SD, 1.5). The most common complication was infection. We found that complication rates were independent of the location of the bone defect. Complication rates in the upper extremity ranged from 0% to 100%, with a total weighted mean of 10%. Conclusion: The induced membrane technique is an emerging possible treatment of large bone defects in the upper extremity. More research is needed to determine the outcomes of the induced membrane technique in the upper extremity.

First Human Leucocyte Antigen (HLA) Response and Safety Evaluation of Fibrous Demineralized Bone Matrix in a Critical Size Femoral Defect Model of the Sprague-Dawley Rat.

Treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. Grafts are necessary to support bone healing. A well-established allograft is demineralized bone matrix (DBM) prepared from donated human bone tissue. In this study, a fibrous demineralized bone matrix (f-DBM) with a high surface-to-volume ratio has been analyzed for toxicity and immunogenicity. f-DBM was transplanted to a 5-mm, plate-stabilized, femoral critical-size-bone-defect in Sprague-Dawley (SD)-rats. Healthy animals were used as controls. After two months histology, hematological analyses, immunogenicity as well as serum biochemistry were performed. Evaluation of free radical release and hematological and biochemical analyses showed no significant differences between the control group and recipients of f-DBM. Histologically, there was no evidence of damage to liver and kidney and good bone healing was observed in the f-DBM group. Reactivity against human HLA class I and class II antigens was detected with mostly low fluorescence values both in the serum of untreated and treated animals, reflecting rather a background reaction. Taken together, these results provide evidence for no systemic toxicity and the first proof of no basic immunogenic reaction to bone allograft and no sensitization of the recipient.

Ex vivo regional gene therapy with human adipose-derived stem cells for bone repair

The treatment of complex bone loss scenarios remains challenging. This study evaluates the efficacy of ex vivo regional gene therapy using transduced human adipose-derived stem cells (ASCs) overexpressing bone morphogenetic protein-2 (BMP-2) to treat critical-sized bone defects. Critical-sized femoral defects created surgically in immunocompromised rats were treated with ASCs transduced with a lentivirus encoding BMP-2 (Group 1, n=14), or green fluorescent protein (Group 2, n=5), nontransduced ASCs (Group 3, n=5), or rhBMP-2 (Group 4, n=14). At 12weeks, femurs were evaluated for quantity and quality of bone formation with plain radiographs, micro-computed tomography, histology/histomorphometry, and biomechanical strength testing. Thirteen of 14 samples in Group 1 and all 14 samples in Group 4 showed radiographic healing, while no samples in either Groups 2 or 3 healed. Groups 1 and 4 had significantly higher radiographic scores (p<0.001), bone volume fraction (BV/TV) (p<0.001), and bone area fraction (BA/TA) than Groups 2 and 3 (p<0.001). Radiographic scores, BV/TV, and BA/TA were not significantly different between Groups 1 and 4. No difference with regards to mean torque, rotation at failure, torsional stiffness, and energy to failure was seen between Groups 1 and 4. Human ASCs modified to overexpress BMP-2 resulted in abundant bone formation, with the quality of bone comparable to that of rhBMP-2. This strategy represents a promising approach in the treatment of large bone defects in the clinical setting. Large bone defects may require sustained protein production to induce an appropriate osteoinductive response. Ex vivo regional gene therapy using a lentiviral vector has the potential to be part of a comprehensive tissue engineering strategy for treating osseous defects.

Introduction of a New Surgical Method to Improve Bone Healing in a Large Bone Defect by Replacement of the Induced Membrane by a Human Decellularized Dermis Repopulated with Bone Marrow Mononuclear Cells in Rat.

The Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. We eliminate the first surgical step by using a decellularized dermal skin graft (Epiflex®) populated with bone marrow mononuclear cells (BMC), as a replacement for the induced membrane. The aim of this study was to demonstrate the feasibility of this technology and provide evidence of equivalent bone healing in comparison to the induced membrane-technique. Therefore, 112 male Sprague–Dawley rats were allocated in six groups and received a 10 mm femoral defect. Defects were treated with either the induced membrane or decellularized dermis, with or without the addition of BMC. Defects were then filled with a scaffold (β-TCP), with or without BMC. After a healing time of eight weeks, femurs were taken for histological, radiological and biomechanical analysis. Defects treated with Epiflex® showed increased mineralization and bone formation predominantly in the transplanted dermis surrounding the defect. No significant decrease of biomechanical properties was found. Vascularization of the defect could be enhanced by addition of BMC. Considering the dramatic reduction of a patient’s burden by the reduced surgical stress and shortened time of treatment, this technique could have a great impact on clinical practice.

Bioactive PLGA/tricalcium phosphate scaffolds incorporating phytomolecule icaritin developed for calvarial defect repair in rat model.

Background/objectivesFor treatment of large bone defects challenging in orthopaedic clinics, bone graft substitutes are commonly used for the majority of surgeons. It would be proposed in the current study that our bioactive scaffolds could additionally serve as a local delivery system for therapeutic small molecule agents capable of providing support to enhance biological bone repair.MethodsIn this study, composite scaffolds made of poly (lactic-co-glycolic acid) (PLGA) and tricalcium phosphate (TCP) named by P/T was fabricated by a low-temperature rapid prototyping technique. For optimizing the scaffolds, the phytomolecule icaritin (ICT) was incorporated into P/T scaffolds called P/T/ICT. The osteogenic efficacies of the two groups of scaffolds were compared in a successfully established calvarial defect model in rats. Bone regeneration was evaluated by X-ray, micro-computerised tomography (micro-CT), and histology at weeks 4 and/or 8 post-implantation. In vitro induction of osteogenesis and osteoclastogenesis was established for identification of differentiation potentials evoked by icaritin in primary cultured precursor cells.ResultsThe results of radiographies and decalcified histology demonstrated more area and volume fractions of newly formed bone within bone defect sites implanted with P/T/ICT scaffold than that with P/T scaffold. Undecalcified histological results presented more osteoid and mineralized bone tissues, and also more active bone remodeling in P/T/ICT group than that in P/T group. The results of histological staining in osteoclast-like cells and newly formed vessels indicated favorable biocompatibility, rapid bioresorption and more new vessel growth in P/T/ICT scaffolds in contrast to P/T scaffolds. Based on in vitro induction, the results presented that icaritin could significantly facilitate osteogenic differentiation, while suppressed adipogenic differentiation. Meanwhile, icaritin demonstrated remarkable inhibition of osteoclastogenic differentiation.ConclusionThe finding that P/T/ICT composite scaffold can enhance bone regeneration in calvarial bone defects through facilitating effective bone formation and restraining excessive bone resorption.The translational potential of this articleThe osteogenic bioactivity of icaritin facilitated PLGA/TCP/icartin composite scaffold to exert significant bone regeneration in calvarial defects in rat model. It might form an optimized foundation for potential clinical validation in bone defects application.

Complications of bone transport technique using the Ilizarov method in the lower extremity: a retrospective analysis of 282 consecutive cases over 10\u2009years

BackgroundThe treatment of large bone defects in lower limbs is a serious challenge for orthopedic surgeons and patients. The bone transport technique using the Ilizarov method has become the main treatment option for the reconstruction of bone defect. However, inevitable difficulties and complications related to bone transport technique have been reported by many studies. The purpose of this study was to evaluate the effectiveness and complications of bone transport technique using Ilizarov method in the treatment of bone defect of lower extremity.MethodsThe study was conducted on 282 patients who underwent bone transport procedures using Ilizarov method at our institution from January 2007 to June 2017. Patient’s demographic data, complications and clinical outcomes at minimum of 2 years follow-up were collected and retrospectively analyzed. All difficulties that related to bone transport were documented according to Paley’s classification. The clinical outcomes were evaluated using Association for the Study and Application of the Method of Ilizarov criteria (ASAMI) at last clinical visit.ResultsThere were 243 male and 39 females with a mean age of 40 years (range 18–65 years). The mean defect was 6.56 ± 2.15 cm, whereas single level transport in 221 cases and double level transport in 61 cases. There were 189 problems, 166 obstacles and 406 complications (257 minor and 149 major complications), and the average complication rate per patients consists of 0.91 minor and 0.53 major complications. The top five complications were pin-site infection (65.96%), axial deviation (40.78%), joint stiffness (23.76%), soft tissue incarceration (22.34%) and delayed union of the docking site (13.48%).The ASAMI bony result was excellent in 233 patients, good in 32, fair in 5 and poor in 12. The ASAMI functional result was excellent in 136 patients, good in 88, fair in 47, poor in 11.ConclusionBone transport is a reliable method for reconstruction of bone defects in the femur and tibia. Awareness of predictable complications is beneficial to prevent or early detection of the expected complication which can improve the risk-benefit balance.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (μCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

High-purity magnesium pin enhances bone consolidation in distraction osteogenesis model through activation of the VHL/HIF-1α/VEGF signaling.

Distraction osteogenesis has widespread clinical use in the treatment of large bone defects. Nonetheless, the prolonged consolidation period carries the risk of complications. Magnesium-based materials have been shown to promote bone regeneration in fracture healing both in vitro and in vivo. Here, we investigated whether high-purity magnesium could enhance bone formation in distraction osteogenesis. High-purity magnesium pins were placed into the medullary cavity in the rat distraction osteogenesis model. Results showed that the bone volume/total tissue volume, bone mineral density, and mechanical properties of new callus were significantly higher in the high-purity magnesium group compared to stainless steel and control group (p < 0.01). Histological analyses confirmed improved bone consolidation and vascularization in high-purity magnesium group. Further, polymerase chain reaction-array investigation, Western blot, and immunohistochemical results found that vascular endothelial growth factor and hypoxia inducible factor-1α were highly expressed in the high-purity magnesium group, while Von Hippel-Lindau protein was the opposite (p < 0.01). In conclusion, high-purity magnesium implants have the potential to enhance angiogenesis and bone consolidation in the distraction osteogenesis application, and this process might be via the regulation of Von Hippel-Lindau/hypoxia inducible factor-1α/vascular endothelial growth factor signaling.

3D-Printing of Hierarchically Designed and Osteoconductive Bone Tissue Engineering Scaffolds.

In Bone Tissue Engineering (BTE), autologous bone-regenerative cells are combined with a scaffold for large bone defect treatment (LBDT). Microporous, polylactic acid (PLA) scaffolds showed good healing results in small animals. However, transfer to large animal models is not easily achieved simply by upscaling the design. Increasing diffusion distances have a negative impact on cell survival and nutrition supply, leading to cell death and ultimately implant failure. Here, a novel scaffold architecture was designed to meet all requirements for an advanced bone substitute. Biofunctional, porous subunits in a load-bearing, compression-resistant frame structure characterize this approach. An open, macro- and microporous internal architecture (100 µm–2 mm pores) optimizes conditions for oxygen and nutrient supply to the implant’s inner areas by diffusion. A prototype was 3D-printed applying Fused Filament Fabrication using PLA. After incubation with Saos-2 (Sarcoma osteogenic) cells for 14 days, cell morphology, cell distribution, cell survival (fluorescence microscopy and LDH-based cytotoxicity assay), metabolic activity (MTT test), and osteogenic gene expression were determined. The adherent cells showed colonization properties, proliferation potential, and osteogenic differentiation. The innovative design, with its porous structure, is a promising matrix for cell settlement and proliferation. The modular design allows easy upscaling and offers a solution for LBDT.

Advances in the Masquelet technique: Myeloid-derived suppressor cells promote angiogenesis in PMMA-induced membranes

The periosteum plays a critical role in bone formation and defect reconstruction. The concept of tissue engineering in the periosteum has been suggested to solve the clinical problems related to bone defect repair. Insertion of polymethyl methacrylate (PMMA) bone cement can induce the autologous generation of a tissue-engineered periosteum and has been considered as a promising strategy for bone defect reconstruction. The PMMA-induced membrane is a crucial element in the reconstruction of bone defects, especially for angiogenesis, but its biological mechanism remains elusive. Here, a PMMA-induced membrane model was established using a femoral critically sized defect in mice. We identified myeloid-derived suppressor cells (MDSCs) as a regulatory component of induced membrane vascularization. The increased number of MDSCs was markedly linked to increased membrane thickness and capillary density. Importantly, the results of an in vitro coculture assay indicated that MDSCs of the induced membrane further facilitated the angiogenic capacity of human umbilical vein endothelial cells (HUVECs) by upregulating the expression of VEGFA, Ang2 and HIF-1α. Furthermore, signaling pathway blockade results suggested that STAT3 activation is involved in the upregulation of VEGFA, Ang2 and HIF-1α expression in induced membrane MDSCs. Our findings provide new insights into the mechanism of angiogenesis in the PMMA-induced membrane and confirm the key signaling molecules of MDSCs in induced membrane angiogenesis. Based on these results, this strategy may become a new therapy for the treatment of large bone defects in the future. Statement of SignificanceIn this study, we established an autologous tissue-engineered periosteum – PMMA-induced membrane, which was formed by the foreign body reaction to PMMA bone cement. The induced membrane establishes a blood supply for the large bone defect healing. After investigation, our study discovered the critical cell type in the formation and angiogenesis processes of the induced membrane, myeloid-derived suppressor cells (MDSCs). We revealed that MDSCs of the induced membrane promote the angiogenesis of endothelial cells through the expression of VEGFA, Ang2 and HIF-1α, which was upregulated by the activation of STAT3 signaling. Our findings clarified the beneficial effect of MDSCs in the angiogenesis of bone repair, and offered an additional target for the study of foreign body reactions to bone repair materials.

From two stages to one: acceleration of the induced membrane (Masquelet) technique using human acellular dermis for the treatment of non-infectious large bone defects

IntroductionThe induced membrane technique for the treatment of large bone defects is a two-step procedure. In the first operation, a foreign body membrane is induced around a spacer, then, in the second step, several weeks or months later, the spacer is removed and the Membrane pocket is filled with autologous bone material. Induction of a functional biological membrane might be avoided by initially using a biological membrane. In this study, the effect of a human acellular dermis (hADM, Epiflex, DIZG gGmbH) was evaluated for the treatment of a large (5 mm), plate-stabilised femoral bone defect.Material and MethodsIn an established rat model, hADM was compared to the two-stage induced membrane technique and a bone defect without membrane cover. Syngeneous spongiosa from donor animals was used for defect filling in all groups. The group size in each case was n = 5, the induction time of the membrane was 3–4 weeks and the healing time after filling of the defect was 8 weeks.ResultsThe ultimate loads were increased to levels comparable with native bone in both membrane groups (hADM: 63.2% ± 29.6% of the reference bone, p < 0.05 vs. no membrane, induced membrane: 52.1% ± 25.8% of the reference bone, p < 0.05 vs. no membrane) and were significantly higher than the control group without membrane (21.5%). The membrane groups were radiologically and histologically almost completely bridged by new bone formation, in contrast to the control Group where no closed osseous bridging could be observed.ConclusionThe use of the human acellular dermis leads to equivalent healing results in comparison to the two-stage induced membrane technique. This could lead to a shortened therapy duration of large bone defects.

Evaluation of Preclinical Models for the Testing of Bone Tissue-Engineered Constructs.

Autologous bone grafting is the clinical gold standard for the treatment of large bone defects, but it can only be obtained in limited amounts and is associated with donor site morbidity. These challenges might be overcome by tissue engineering (TE). Although promising results have been reported, translation into clinics often fails. Lack of reproducibility in preclinical studies may be one of the reasons. We evaluated preclinical models for testing of novel TE strategies, as well as the perception of researchers and clinicians toward the models. Therefore, a review of publications on preclinical models of the past 10 years was performed. A survey addressed to both clinicians and scientists was conducted to assess the clinical need for bone tissue engineering (BTE) constructs and researchers were asked about their satisfaction with the currently available preclinical models. A literature review revealed 169 articles on in vivo studies in the field of BTE, including 26 studies utilizing large animal models and 143 studies in small animals, with rabbits and rats presenting the most commonly used species. Only a few studies used skeletally mature animals, which is in large contrast to the patients targeted. The localization of the bone defects varied, but the vast majority (60%) were segmental bone defects with various fixation techniques. Results of 70 surveys confirmed a great clinical need for TE constructs and positive perceptions of all participants toward its future clinical application. Nevertheless, the need for optimization of preclinical models and limitations when it comes to translation of results to the clinical situation were indicated. No clear trends were detected with regards to the preclinical model, leading to most satisfying results despite the trend that scientists rated generally large animal models higher than small animal models. Results of the literature review and the survey reveal the lack of standardized methods. Despite the affirmed clinical need as well as a very positive perception of clinicians toward the use of TE, results indicate a critical need to optimize preclinical models and, in particular, improve translational aspects of the models. A consensus in the field on a limited number of well-standardized models should be reached.

Femoral bone defect healing using two novel biocompatible degradable materials

This study was conducted as anin vivoexperiment in adult miniature pigs with the aim to test two new biomaterials. An iatrogenic defect was made into the central femoral diaphysis in the experimental animals and subsequently fixated by bridging plate osteosynthesis. Into the defect we implanted a cancellous autograft (control group), a pasty injectable scaffold (EXP A), and a porous 3D cylinder (EXP B). Radiological examination was performed in all animals at 0, 10, 20, 30 weeks after surgical procedure and histological assessment was performed. In the newly formed bone the osteoblastic activity was monitored. In terms of radiology, the most effective method was observed in the control group (completely healed 100%) compared to experimental groups EXP A (70.0%) and EXP B (62.5%). Histological assessment showed a higher cell count in the place of bone defect in the control group compared to experimental groups. Between the experimental groups, a higher count of bone marrow cells was found in group EXP B. Both newly developed biomaterials seem to be suitable as replacements for large bone defects, having good workability and applicability. However, compared to the control group treated with a cancellous autograft, the newly formed bone did not reach the same number of cells settling in and in some cases, full radiological healing was not reached. Nevertheless, the material was found to be grown into the original bone in all cases within the experimental groups. The new biomaterials have a great potential as a substitute in the treatment of large bone defects.

Cell Therapy: Effect of Locally Injected Mesenchymal Stromal Cells Derived from Bone Marrow or Adipose Tissue on Bone Regeneration of Rat Calvarial Defects

Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.

Outcomes of Vascularized Bone Allotransplantation with Surgically Induced Autogenous Angiogenesis in a Large Animal Model: Bone Healing, Remodeling, and Material Properties.

Bone vascularized composite allotransplantation (VCA) is a possible alternative for the treatment of large bone defects. Clinical application of VCAs is limited by the need for life-long immunosuppression (IS). We report an alternative method to maintain bone allotransplant viability in a large animal model without the need for life-long IS by using autogenous vessel implantation. Fourteen bone only VCAs were transplanted in a porcine tibia defect model with short-term IS. Two groups were used to evaluate the effect of the implantation of an autogenous arteriovenous (AV)-bundle, therefore the only difference between the groups was the patency of the AV-bundle. We radiographically evaluated bone healing and allogenic pedicle patency. AV-bundle patency and union were evaluated with micro-CT. Bone remodeling was assessed with histomorphometry and material properties were evaluated with axial compression testing and cyclic reference point indentation. Two subjects did not reach the final time point. Twelve tibiae healed proximally, and nine at the distal transplant-bone interface. Bone allotransplants showed their viability in the first 4 to 6 weeks by significant periosteal bridging arising from the transplant and maintained pedicle patency. Bone material properties were not affected by the implantation of an AV-bundle when compared with ligated AV-bundle controls, but diminished compared with normal bone. Significantly higher bone formation rates resulted from the implantation of a patent AV-bundle. New periosteal bone formation and subsequent bone healing result from blood flow through the microsurgically repaired nutrient blood supply, demonstrated by maintained allogenic pedicle patency. The implantation of a patent autogenous AV-bundle has no adverse effect on material properties, but a positive effect on bone remodeling of endosteal surfaces despite thrombosis of the allogenic pedicle. Bone material properties change after transplantation compared with normal bone, although 20-weeks survival time is relatively short for the final evaluation of bone material properties.