Summary Statement

Supercritical fluid (SCF) sterilization produces clean and osteoconductive allograft bone capable of healing a critical-sised bony defect. SCF treated graft induces an increased anabolic response and decreased catabolic reponse compared to gamma irradiated graft.

The need for an artificial scaffold in very large bone defects is clear, not only to limit the risk of graft harvesting, but also to improve clinical success. The use of custom osteoconductive scaffolds made from biodegradable polyester and ceramics can be a valuable patient friendly option, especially in case of a concomitant infection. Multiple types of scaffolds for the Masquelet procedure (MP) are available, however these frequently demonstrate central graft involution when defects exceed a certain size and the complication rates remains high. This paper describes three infected tibial defect nonunions with a segmental defect over ten centimeters long treated with a customized 3D printed polycaprolactone-tricalcium phosphate (PCL-TCP) cage in combination with biological adjuncts. Three male patients, between the age of 37 and 47, were treated for an infected tibial defect nonunion after sustaining Gustilo grade 3 open fractures. All had a segmental midshaft bone defect of more than ten centimeters (range 11–15cm). First stage MPs consisted of extensive debridement, external fixation and placement of anterior lateral thigh flaps (ALT). Positive cultures were obtained from all patients during this first stage, that were treated with specific systemic antibiotics during 12 weeks. The second stage MP was carried out at least two months after the first stage. CT scans were obtained after the first stage to manufacture defect-specific cages. In the final procedure a custom 3D printed PCL-TCP cage (Osteopore, Singapore) was placed in the defect in combination with biological adjuncts (BMAC, RIA derived autograft, iFactor and BioActive Glass). Bridging of the defect, assessed at six months by CT, was achieved in all cases. SPECT-scans 6 months post-operatively demonstrated active bone regeneration, also involving the central part of the scaffold. All three patients regained function and reported less pain with full weight-bearing. This case report shows that 3D printed PCL-TCP cages in combination with biological adjuncts is a novel addition to the surgical treatment of very large bone defects in (infected) posttraumatic nonunion of the tibia. This combination could overcome some of the current drawbacks in this challenging indication

Our previous rat study demonstrated an ex vivo-created “Biomimetic Hematoma” (BH) that mimics the intrinsic structural properties of normal fracture hematoma, consistently and efficiently enhanced the healing of large bone defects at extremely low doses of rhBMP-2 (0.33 μg). The aim of this study was to determine if an extremely low dose of rhBMP-2 delivered within BH can efficiently heal large bone defects in goats. Goat 2.5 cm tibial defects were stabilized with circular fixators, and divided into groups (n=2-3): 2.1 mg rhBMP-2 delivered on an absorbable collagen sponge (ACS); 52.5 μg rhBMP-2 delivered within BH; and an empty group. BH was created using autologous blood with a mixture of calcium and thrombin at specific concentrations. Healing was monitored with X-rays. After 8 weeks, femurs were assessed using microCT. Using 2.1 mg on ACS was sufficient to heal 2.5 cm bone defects. Empty defects resulted in a nonunion after 8 weeks. Radiographic evaluation showed earlier and more robust callus formation with 97.5 % (52.5 μg) less of rhBMP-2 delivered within the BH, and all tibias were fully bridged at 3 weeks. The bone mineral density was significantly higher in defects treated with BH than with ACS. Defects in the BH group had smaller amounts of intramedullary and cortical trabeculation compared to the ACS group, indicating advanced remodeling. The results confirm that the delivery of rhBMP-2 within the BH was much more efficient than on an ACS. Not only did the large bone defects heal consistently with a 40x lower dose of rhBMP-2, but the quality of the defect regeneration was also superior in the BH group. These findings should significantly influence how rhBMP-2 is delivered clinically to maximize the regenerative capacity of bone healing while minimizing the dose required, thereby reducing the risk of adverse effects

Background. Tissue engineering strategies to heal critical-size bone defects through direct bone formation are limited by incomplete integration of grafts with host bone and incomplete vascularisation. An alternative strategy is the use of cartilage grafts that undergo endochondral ossification. Endochondral cartilages stimulate angiogenesis and are remodeled into bone, but are naturally found in only small quantities. We sought to develop engineered endochondral cartilage grafts using human osteoarthritic (OA) articular chondrocytes. Methods. Study approval was obtained from our human and animal ethics review committees. Human OA cartilage was obtained from discarded tissues from total knee replacements. Scaffold-free engineered grafts were generated by pelleting primary or passaged chondrocytes, followed by culture with transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4. Samples were transplanted into immunocompromised mice either subcutaneously or into critical-size tibial defects. Grafts derived from passaged chondrocytes from either of two patients (64 year old and 68 year old men) where implanted into tibial defects in five mice. Bone formation was assessed with histology after four weeks of implantation. Results. Engineered cartilage grafts generated from passaged OA chondrocytes underwent endochondral ossification after implantation either subcutaneously or in bone. The grafts bridged tibial defects, integrating with bone proximally and distally in all cases. Portions of the graft were remodeled into woven bone, which spanned the defects in two animals. Unmodified OA cartilage and engineered grafts formed from primary chondrocytes did not undergo endochondral ossification in vivo. Conclusions. Human OA chondrocytes adopt an endochondral phenotype after passaging and TGF-β superfamily treatment. Engineered endochondral cartilage grafts can integrate with host bone, undergo ossification, and heal critical-size long-bone defects in a mouse model. Level of Evidence. Animal study. Disclosure. A patent application on this technology has been filed

Aims. Metaphyseal tritanium cones can be used to manage the tibial bone loss commonly encountered at revision total knee arthroplasty (rTKA). Tibial stems provide additional fixation and are generally used in combination with cones. The aim of this study was to examine the role of the stems in the overall stability of tibial implants when metaphyseal cones are used for rTKA. Methods. This computational study investigates whether stems are required to augment metaphyseal cones at rTKA. Three cemented stem scenarios (no stem, 50 mm stem, and 100 mm stem) were investigated with 10 mm-deep uncontained posterior and medial tibial defects using four loading scenarios designed to mimic activities of daily living. Results. Small micromotions (mean < 12 µm) were found to occur at the bone-implant interface for all loading cases with or without a stem. Stem inclusion was associated with lower micromotion, however these reductions were too small to have any clinical significance. Peak interface micromotion, even when the cone is used without a stem, was too small to effect osseointegration. The maximum difference occurred with stair descent loading. Stress concentrations in the bone occurred around the inferior aspect of each implant, with the largest occurring at the end of the long stem; these may lead to end-of-stem pain. Stem use is also found to result in stress shielding in the bone along the stem. Conclusion. When a metaphyseal cone is used at rTKA to manage uncontained posterior or medial defects of up to 10 mm depth, stem use may not be necessary. Cite this article:Bone Joint Res. 2020;9(4):162–172

Metaphyseal fracture healing is important in joint-adjacent fractures and appears to differ from diaphyseal healing. We recently found that a biomaterial delivering bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA) healed the metaphyseal bone in a tibial defect but failed closing the cortical defect. In this study we added a BMP-2 soaked collagen membrane to study cortical healing from the muscle tissue surrounding the bone. We used SD rats and a 4.5 mm metaphyseal circular tibial defect. In group 1 (G1), a porous gelatin-calcium sulphate-hydroxyapatite (GCH) biomaterial containing rhBMP-2 and ZA was used to fill the defect (GCH+5 μg BMP-2+10 μg ZA). In group 2 (G2), we used a collagen membrane (2 μg BMP-2) to cover the GCH filled defect (GCH+3μg BMP+10 μg ZA). Group 3 (G3) was an empty control. Animals were sacrificed after 8-weeks and bone regeneration was evaluated with micro-CT and histology. In both G1 (P<0.001) and G2 (p<0.001) a significantly higher mineralized volume was found in the defect compared to empty G3. In G2 higher mineralized volume was found in the cortical region compared to both G1 (p<0.01) and G3 (p<0.001) as seen via micro-CT. Histologically, G1 and G2 showed islands of trabecular bone in the defect peripherally but only G2 showed cortical healing. G3 was empty in the middle but showed healed cortex. In conclusion, GCH can be used to deliver BMP-2 and ZA to promote metaphyseal bone growth. A membrane (CM) doped with low dose BMP-2 improved cortical regeneration

Summary Statement. Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HA. Glu. ) improves the bone repair in a cavitary defect in rat tibiae. Introduction. Biopolymer-coated nanocrystalline hydroxyapatite (HA. Glu. ) made as macroporous foams are promising candidates as scaffolds for bone tissue engineering applications. They exhibit optimal features, promoting internalization, proliferation and differentiation of osteoprogenitors, with an adequate cell colonization over the entire scaffold surface. Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Its 107–111 epitope (osteostatin) exhibits osteogenic properties at least in part by directly acting on osteoblasts. The main aim of this study was to evaluate whether osteostatin loading into HA. Glu. scaffolds might improve their bone regeneration capacity. Materials and Methods. HA. Glu. scaffolds were prepared as previously described (Sánchez-Salcedo S et al. J. Mater. Chem. 2010; 20:6956-61). Osteostatin was adsorbed onto HA. Glu. material by dipping into a solution containing this peptide at 100 nM (in phosphate-buffered saline, pH 7.4), following a standard protocol. We performed a cavitary defect (2 mm in diameter and 3 mm in depth) in both distal tibial metaphysis using a drill under general anesthesia in male Wistar rats (n=8) of 6 months of age. Unloaded HA. Glu. material (7 mg) was implanted into left tibial defects, whereas rigth tibial defects received the osteostatin-loaded material. Animals were sacrificed after 4 weeks for histological, μ-computerised tomography and gene expression analysis of the callus. Our protocol was approved by the Institutional Animal Care and Use Committee at the IIS-FJD. Mouse osteoblastic MC3T3-E1 cells were grown in differentiation medium (α-MEM with 10% fetal bovine serum, 50 µg/ml ascorbic acid, and 10 mM β-glycerolphosphate), in the presence or absence of HA. Glu. material with or without osteostatin. Cell viability (assessed by trypan blue staining), alkaline phosphatase (ALP) activity and mineralization (alizarin red) were analyzed at different culture times. Results. The mean uptake of osteostatin by HA. Glu. scaffolds was about 60 % (representing 0.7 μg/implanted scaffold) after 24 h of loading, and they released a mean of 80 % of loaded peptide to the surrounding medium within 1–24 h. At 4 weeks, this osteostatin-containing HA. Glu. material significantly increased the bone volumen fraction and trabecular thickness of regenerating bone in the tibial methaphysis, compared to those observed with unloaded HAGlu scaffolds. In addition, osteostatin-coated HA. Glu. scaffolds increased (2-fold) the gene expression of osteocalcin and vascular cell adhesion molecule 1, but decreased (2-fold) that of the Wnt inhibitors, SOST and Dickkopf homolog 1 (DKK-1) in the fracture callus. In MC3T3-E1 cell cultures, osteostatin-loaded HA. Glu. material increased cell viability and ALP activity (each by 30%), and matrix mineralization (by 50%) at days 4 and 10, respectively. Conclusions. These results indicate that osteostatin loading improves the bone regeneration capacity of HA. Glu. scaffolds. Our findings suggest that these scaffolds might be promising implants in orthopaedic applications. This work has been supported by a grant from Comunidad Autónoma de Madrid (S-2009/MAT/1472)

The treatment of bony defects of the tibia at the time of revision total knee replacement is controversial. The place of compacted morsellised bone graft is becoming established, particularly in contained defects. It has previously been shown that the initial stability of impaction-grafted trays in the contained defects is equivalent to that of an uncemented primary knee replacement. However, there is little biomechanical evidence on which to base a decision in the treatment of uncontained defects. We undertook a laboratory-based biomechanical study comparing three methods of graft containment in segmental medial tibial defects and compared them with the use of a modular metal augment to bypass the defect. Using resin models of the proximal tibia with medial defects representing either 46% or 65% of the medial cortical rim, repair of the defect was accomplished using mesh, cement or a novel bag technique, after which impaction bone grafting was used to fill the contained defects and a tibial component was cemented in place. As a control, a cemented tibial component with modular metal augments was used in identical defects. All specimens were submitted to cyclical mechanical loading, during which cyclical and permanent tray displacement were determined. The results showed satisfactory stability with all the techniques except the bone bag method. Using metal augments gave the highest initial stability, but obviously lacked any potential for bone restoration

Background. Bio-Active Glass (BAG) is a promising bone graft substitute for large bone defect reconstruction because of its favourable osteoconductive, antibacterial and angiogenic properties. Potentially, it could also mechanically reinforce the defect, thus making it suitable for load-bearing defects. However, the mechanical properties of the reconstructive layer consisting of BAG/bone allograft mixtures are unknown. The goals of this study therefore were, first, to measure the mechanical properties of different BAG/bone graft mixtures and, second, to investigate to what extent such mixtures could reinforce distal tibial defects using micro-FE analysis and high-resolution CT scans. Materials and Methods. Four different BAG/bone graft mixtures were impacted in a cylindrical holder, mechanically tested in confined compression and scanned with micro-CT. From these images, bone graft material and glass were segmented using two different threshold values. The interface between bone and BAG was modelled separately by dilating the glass phase. Micro-Finite-Element (FE) models of the composites were made using a Young's modulus of 2.5 GPa for bone and 35 GPa for BAG. The Young's modulus for the interface region was determined by fitting experimental and micro-FE results for the same specimens. (82 μm resolution) CT scans of a 9 mm region of the distal tibia of 3 subjects were used. Micro-FE models of this region were made to determine its stiffness in the original state, with a simulated cortical defect and after a mixture of BAG/bone was modelled in the defect. Results. The confined compression tests showed a strong dependence of the modulus of the BAG/bone composite on the amount of BAG, ranging from 116.7 ± 18.2 to 654.2 ± 35.2 MPa. The micro-FE results could well reproduce these measured moduli, when using a stiffness of 25 MPa for the interface layer. The micro-FE analyses of the cortical defect demonstrated that the stiffness of the tibial segment would be reduced by 13 ± 3 % with the defect. Treatment with the BAG/bone composite could restore the stiffness to 101 ± 6 % of its original value. Discussion. The experiments demonstrate that BAG/bone mixtures have a composition-dependent stiffness, in the range of that of trabecular bone, which can be well estimated from micro-FE analyses. Furthermore, the tibial micro-FE analyses demonstrate that these mixtures potentially can restore the stiffness of large bone defects at this site. Future development of the model may predict mechanical behaviour of BAG/bone mixtures patient specifically

Objectives

We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells.

Critical size defects in ovine tibiae, stabilised with intramedullary interlocking nails, were used to assess whether the addition of carboxymethylcellulose to the standard osteogenic protein-1 (OP-1/BMP-7) implant would affect the implant’s efficacy for bone regeneration. The biomaterial carriers were a ‘putty’ carrier of carboxymethylcellulose and bovine-derived type-I collagen (OPP) or the standard with collagen alone (OPC). These two treatments were also compared to “ungrafted” negative controls. Efficacy of regeneration was determined using radiological, biomechanical and histological evaluations after four months of healing. The defects, filled with OPP and OPC, demonstrated radiodense material spanning the defect after one month of healing, with radiographic evidence of recorticalisation and remodelling by two months. The OPP and OPC treatment groups had equivalent structural and material properties that were significantly greater than those in the ungrafted controls. The structural properties of the OPP- and OPC-treated limbs were equivalent to those of the contralateral untreated limb (p > 0.05), yet material properties were inferior (p < 0.05). Histopathology revealed no residual inflammatory response to the biomaterial carriers or OP-1. The OPP- and OPC-treated animals had 60% to 85% lamellar bone within the defect, and less than 25% of the regenerate was composed of fibrous tissue. The defects in the untreated control animals contained less than 40% lamellar bone and more than 60% was fibrous tissue, creating full cortical thickness defects. In our studies carboxymethylcellulose did not adversely affect the capacity of the standard OP-1 implant for regenerating bone.