Introduction: The mechanobiology and response of bone formation to strain under physiological loading is well established, however investigation into exceedingly soft scaffolds relative to cancellous bone is limited. In this study we designed and 3D printed mechanically-optimised low-stiffness implants, targeting specific strain ranges inducing bone formation and assessed their biological performance in a pre-clinical in vivo load-bearing tibial tuberosity advancement (TTA) model. The TTA model provides an attractive pre-clinical framework to investigate implant osseointegration within an uneven loading environment due to the dominating patellar tendon force. A knee finite element model from ovine CT data was developed to determine physiological target strains from simulated TTA surgery. We 3D printed low-stiffness Ti wedge osteotomy implants with homogeneous stiffness of 0.8 GPa (Ti1), 0.6 GPa (Ti2) and a locally-optimised design with a 0.3 GPa cortex and soft 0.1 GPa core (Ti3), for implantation in a 12-week ovine tibial advancement osteotomy (9mm). We quantitatively assessed bone fusion, bone area, mineral apposition rate and bone formation rate. Optimised Ti3 implants exhibited evenly high strains throughout, despite uneven wedge osteotomy loading. We demonstrated that higher strains above 3.75%, led to greater bone formation. Histomorphometry showed uniform bone ingrowthin optimised Ti3 compared to homogeneous designs (Ti1 and Ti2), and greater bone-implant contact. The greatest bone formation scores were seen in Ti3, followed by Ti2 and Ti1. Results from our study indicate lower stiffness and higher strain ranges than normally achieved in Ti scaffolds stimulate early bone formation. By accounting for loading environments through rational design, implants can be optimised to improve uniform osseointegration. Design and 3D printing of exceedingly soft titanium orthopaedic implants enhance strain induced bone formation and have significant importance in future implant design for knee, hip arthroplasty and treatment of large load-bearing bone defects

Polyetheretherketone (PEEK) interbody fusion cages combined with autologous bone graft is the current clinical gold standard treatment for spinal fusion, however, bone graft harvest increases surgical time, risk of infection and chronic pain. We describe novel low-stiffness 3D Printed titanium interbody cages without autologous bone graft and assessed their biological performance in a pre-clinical in vivo interbody fusion model in comparison to the gold standard, PEEK with graft. Titanium interbody spacers were 3D Printed with a microporous (Ti1: <1000μm) and macroporous (Ti2: >1000μm) design. Both Ti1 and Ti2 had an identical elastic modulus (stiffness), and were similar to the elastic modulus of PEEK. Interbody fusion was performed on L2-L3 and L4-L5 vertebral levels in 24 skeletally mature sheep using Ti1 or Ti2 spacers, or a PEEK spacer filled with iliac crest autograft, and assessed at 8 and 16 weeks. We quantitatively assessed bone fusion, bone area, mineral apposition rate and bone formation rate. Functional spinal units were biomechanically tested to analyse range of motion, neutral zone, and stiffness. Results: Bone formation in macroporous Ti2 was significantly greater than microporous Ti1 treatments (p=.006). Fusion scores for Ti2 and PEEK demonstrated greater rates of bone formation from 8 to 16 weeks, with bridging rates of 100% for Ti2 at 16 weeks compared to just 88% for PEEK and 50% for Ti1. Biomechanical outcomes significantly improved at 16 versus 8 weeks, with no significant differences between Ti and PEEK with graft. This study demonstrated that macroporous 3D Printed Ti spacers are able to achieve fixation and arthrodesis with complete bone fusion by 16 weeks without the need for bone graft. These significant data indicate that low-modulus 3D Printed titanium interbody cages have similar performance to autograft-filled PEEK, and could be reliably used in spinal fusion avoiding the complications of bone graft harvesting

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Aim. Antibiotic-loaded biomaterials are often used in dead space management after excision of infected bone. This study assessed the chronological progression of new bone formation in infected defects, filled only with an absorbable, osteoconductive bone void filler with Gentamicin (1). Method. 163 patients were treated for osteomyelitis or infected fractures with a single-stage excision, implantation of antibiotic carrier, stabilisation and wound closure. All had Cierny & Mader Type III (n=128) or Type IV (n=35) infection. No bone grafting was performed in any patient. Patients were followed up for a minimum of 12 months (mean 21.4 months; 12–56). Bone void filling was assessed on serial digitised, standardized radiographs taken immediately after surgery, at 6 weeks, 3, 6 and 12 months and then yearly. Data on defect size, location, degree of void filling, quality of the bone-biomaterial interface and material leakage were collected. Bone formation was calculated at final follow-up, as a percentage of initial defect volume, by determining the bone area on AP and lateral radiographs to the nearest 5%. Results. 138 patients had adequate radiographs for assessment. Infection was eradicated in 95.7%. 2.5% of patients suffered a fracture during follow-up. Overall, bone formation was good (mean 73.8% defect filling), with one quarter of patients having complete defect filling and 87% having more than 50% of the defect healed. Bone formed better in metaphyseal defects compared to diaphyseal defects (mean 79% filling vs 66%; p<0.02). Good interdigitation of the biomaterial with the host bone, seen on the initial radiograph, was associated with more bone formation (77% vs 69%; p=0.021). Leakage of the biomaterial out of the defect reduced mean bone formation from 77% to 62% (p=0.006). 38 cases had radiographs more than 2 years after implantation. In 24 (63.2%), bone formation continued to increase after the first year radiograph. Conclusion. This biomaterial was effective in allowing significant amounts of bone to form in the defect. This removed the need for bone grafting in this series, with a low risk of fracture or recurrent infection. However, bone formation is affected by the site of the lesion and the adequacy of filling at surgery. It is important to achieve good contact between the bone surface and the biomaterial at operation. Bone formation is slow and progresses for at least 2 years after implantation, in two thirds of patients

Purpose. Vitamin D is a key regulator of bone homeostasis. The enzyme CYP24A1 is responsible for transforming vitamin D into 24,25(OH)2vitD. The putative biological activity of 24,25(OH)2vitD remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following a fracture in chicks. Our laboratory has engineered a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25(OH)2vitD. We set out to study the role of 24,25(OH)2vitD in endochondral and intramembranous bone formation in fracture repair in this mouse model based on the results of the chick fracture repair study. Method. Wild-type and mutant Cyp24a1 gene deficient mice were subjected to two different surgical procedures to simulate bone development and fracture repair. To mimic endochondral ossification, we devised a modified technique to perform intramedullary nailing of a mouse tibia followed by an induced fracture. To evaluate intramembranous ossification, we applied distraction osteogenesis to a mouse tibia using a mini Ilizarov external fixator apparatus. Histomorphometric parameters and gene expression differences in fracture repair between the mutant mice and the wild-type controls were measured using micro computed tomography, histology and reverse-transcription quantitative PCR (RT-qPCR) respectively. Results. Quantitative histomorphometric results showed a delay in endochondral fracture repair in the mutant mice calluses as compared to the wild-type mice calluses. In the same model, gene expression of type X collagen in the callus was higher in the wild-type mice. These significant differences were fully rescued by injecting the mutant mice with exogenous 24,25(OH)2vitD. In the intramembranous bone formation model, we found a trend towards reduced bone formation in the gap created by the distraction process in the mutant mice as compared to the wild-type mice. However, the differences did not reach statistical significance. Conclusion. Our results support a role for 24,25(OH)2vitD in fracture repair which is more dominant in a chondrocyte-mediated bone formation pathway like endochondral ossification. Although our results did not reach statistical significance in the intramembranous ossification model, the observed trend suggests a potential role as well. Further study of the role of 24,25(OH)2vitD in bone healing has the potential to support novel approaches in accelerating bone formation and fracture repair

Bone morphogenetic proteins (BMPs) are able to induce osteogenic differentiation in many cells, including muscle cells. However, the actual contribution of muscle cells to bone formation and repair is unclear. Our objective was to examine the capacity of myogenic cells to contribute to BMP-induced ectopic bone formation and fracture repair. Osteogenic gene expression was measured by quantitative PCR in osteoprogenitors, myoblasts, and fibroblasts following BMP-2 treatment. The MyoD-Cre x ROSA26R and MyoD-Cre x Z/AP mouse strains were used to track the fate of MyoD+ cells in vivo. In these double-transgenic mice, MyoD+ progenitors undergo a permanent recombination event to induce reporter gene expression. Ectopic bone was produced by the intramuscular implantation of BMP-7. Closed tibial fractures and open tibial fractures with periosteal stripping were also performed. Cellular contribution was tracked at one, two and three week time points by histological staining. Osteoprogenitors and myoblasts exhibited comparable expression of early and late bone markers; in contrast bone marker expression was considerably less in fibroblasts. The sensitivity of cells to BMP-2 correlated with the expression of BMP receptor-1a (Bmpr1a). Pilot experiments using the MyoD-Cre x Rosa26R mice identified a contribution by MyoD expressing cells in BMP-induced ectopic bone formation. However, false positive LacZ staining in osteoclasts led us to seek alternative systems such as the MyoD-cre x Z/AP mice that have negligible background staining. Initially, a minor contribution from MyoD expressing cells was noted in the ectopic bones in the MyoD-cre x Z/AP mice, but without false positive osteoclast staining. Soft tissue trauma usually precedes the formation of ectopic bone. Hence, to mimic the clinical condition more precisely, physical injury to the muscle was performed. Traumatising the muscle two days prior to BMP-7 implantation: (1) induced MyoD expression in quiescent satellite cells; (2) increased ectopic bone formation; and (3) greatly enhanced the number of MyoD positive cells in the ectopic bone. In open tibial fractures the majority of the initial callus was MyoD+ indicating a significant contribution by myogenic cells. In contrast, closed fractures with the periosteum intact had a negligible myogenic contribution. Myoblasts but not fibroblasts were highly responsive to BMP stimulation and this was associated with BMP receptor expression. Our transgenic mouse models demonstrate for the first time that muscle progenitors can significantly contribute to ectopic bone formation and fracture repair. This may have translational applications for clinical orthopaedic therapies

Objective. Superior bone ingrowth and resistance to bacterial infection are ideal for orthopaedic implants. We compared new bone formation, strength of bone bonding, and infection rates between silicon nitride ceramic (Si3N4; abbreviated SiN), medical-grade PEEK (PEEK), and titanium (Ti) in rat calvariae. PEEK and Ti are used in spinal and arthroplasty implants respectively, while SiN is a non-oxide ceramic used in spinal implants for the past 4 years. Methods. Specimens of 10 mm × 10 mm by 1.75 mm size were implanted into experimental calvarial defects in 2-year old Wistar rats using standard surgical techniques (n's: SiN=48; PEEK=24; Ti=24). One group of animals was immediately inoculated with 1 × 104 Staphylococcus epidermidis; control animals received saline only. After sacrifice at 3 days, 7 days, 14 days, and 3 months post-inoculation (n=4 rats per time period), one calvarial sample each for PEEK and Ti, and two samples for SiN (per bacterial condition and time point) were retrieved for histology; remaining samples were used for sample push-out testing with a Micro Tester 5848 (Instron) with a 1-kN load cell, using published techniques. New bone formation was measured with tetracycline double-labeling at 11 and 4 days before the 14-day and 3-month time periods. Results. Of the materials tested, 3-month bone ingrowth and periprosthetic infection rates were most favorable for SiN (Fig. 1). At 90 days post-implantation without bacteria, new bone growth in calvariae with SiN was ∼69% compared with 24% and 36% for PEEK and Ti, respectively. With bacterial inoculation, new bone growth was 21%, 26% and 41% for PEEK, Ti, and SiN, respectively. At 3-months, live bacteria were observed for PEEK (88%) and Ti (21%), while no bacteria were present around SiN (Fig. 2). Push-out strength was greater for SiN when compared to Ti and PEEK (Fig. 3). Conclusions. Superior bone formation, bone ingrowth, and bacterial resistance were associated with SiN when compared to Ti and PEEK. SiN proved effective in inhibiting bacteria and promoting osteogenesis in experimental osseous defects, when compared to Ti and PEEK. These observations are most likely related to the hydrophilic properties of SiN that contributed to the adsorption of proteins known to decrease bacteria attachment and growth (vitronectin and fibronectin). We conclude that SiN may be a superior biomaterial for the development of orthopaedic implants

Aim

In this study we investigated the effects of non-steroidal anti-inflammatory drugs (NSAIDs) with different cyclooxygenase (COX) selectivity on orthopaedic device-related infections (ODRIs) in a rat model. Specifically, we aimed to measure the impact of NSAID therapy on bone changes, bacterial load, and cytokine levels after treatment with antibiotics. In addition, we compared the effects of long vs short-term celecoxib (a COX-2 inhibitor) treatment on the same outcomes.

We investigated the effects of non-steroidal anti-inflammatory drugs (NSAIDs) with different cyclooxygenase (COX) selectivity on orthopaedic device-related infections (ODRIs) in a rat model. We aimed to measure the impact of NSAID therapy on bone changes, bacterial load, and cytokine levels after treatment with antibiotics. We also compared the effects of long vs short-term celecoxib (a COX-2 inhibitor) treatment on the same outcomes.

Skeletally mature female Wistar rats were implanted with Staphylococcus epidermidis- contaminated polyetheretherketone (PEEK) screws in the proximal right tibia and monitored for 7 days. All animals received subcutaneous antibiotics (rifampicin plus cefazolin) for two weeks from day 7 to 21. In phase I of the study, rats were randomly assigned to receive 28 days of oral treatment with acetylsalicylic acid, ibuprofen, celecoxib, or vehicle control. In phase II, an additional group received seven days of celecoxib treatment from day 0 to 7. Bone changes were monitored using in vivo micro-CT and histology. Quantitative bacteriology was performed at euthanasia. Plasma samples were collected to measure cytokine levels on days 0, 6, 20, and 28.

Combination antibiotic therapy resulted in treatment success in 85.71% of cases, while the addition of long-term celecoxib treatment reduced it to 45.45%. Long-term celecoxib treatment significantly reduced bone loss (33.85% mean difference [95% CI 14.12–53.58], p=0.0004 on day 6 bone fraction) and periosteal reaction (0.2760 μm mean difference [95% CI 0.2073–0.3448], p<0.0001 on day 14 periosteal thickness) during early infection compared to the control group. Short- term celecoxib treatment showed similar radiological results without a reduction in treatment success (88.9%). No differences in the inflammatory markers were observed.

Our findings highlight the potential benefits of short-term use of celecoxib in improving bone fraction during the early post-infection period without impairing the efficacy of antibiotic therapy

To test the hypothesis that: CERAMENT[™]|G (C-G) would improve new bone growth and decrease infection rate after debridement as compared with 1) CERAMENT|BONE VOID FILLER (CBVF) and 2) no void filler in a rat osteomyelitis model.

72 Sprague Dawley rats were injected with 1.5 × 10∧6 CFU of S. aureus into a drill hole in the right tibia. After 3 weeks, the osteomyelitic defect was debrided, and filled with either: 1) C-G (n=32), 2) CBVF (n=20), or 3) nothing (n=20). 6 weeks after the second surgery, 20 rats from each group were sacrificed and the right tibias were harvested. A long-term group (n=12) of C-G treated rats were also sacrificed at 6 months after the second surgery. The tissues were sonicated and the colony forming units in the sonicate were quantified by serial dilutions and culture. MicroCT was used to quantify the new bone growth (BV/TV) in the debrided osteomyelitic void. Histological samples were analyzed for the presence of a neutrophil response by a blinded pathologist.

(*: p<0.05)

Positive cultures in:

○ 30% of animals treated with CBVF

Neutrophil reaction in:

○ 35% of animals treated with CBVF

The BV/TV in:

○ C-G treated rats was 24% greater than CBVF treated rats (*)

Animals sacrificed at 6 months which were treated with C-G did not have any evidence of infection by culture or histology. The bone mass of the implanted limb was higher than the contralateral (non-operated) side.

CERAMENT|G decreased the rate of infection and increased new bone growth as compared with both CBVF and no void filler in a debrided osteomyelitic environment. Animals treated with C-G at 6 months showed no evidence of infection and retained a higher bone mass relative to the contralateral (non-operated) side.

This study supports the use of CERAMENT|G as a readily available void filler which could be used in osteomyelitic environments after debridement.

Custom 3D printed implants can be anatomically designed to assist in complex surgery of the bony pelvis in both orthopaedic oncology and orthopaedic reconstruction surgery. This series includes patients who had major pelvic bone loss after initially presenting with tumours, fractures or infection after previous total hip arthroplasty. The extent of the bone loss in the pelvis was severe and therefore impossible to be reconstructed by conventional ‘off –the-shelve’ implants. The implant was designed considering the remaining bony structures of the contra-lateral hemi- pelvis, to provide an anatomical, secured support for the reconstructed hip joint. The latter was realised by strategically orientated screws and by porous structures (an integral part of the implant), which stimulates osseointegration. A custom pelvic implant was designed, manufactured and 3D printed. Reconstruction of the pelvis was performed together with a cemented (bipolar bearing) acetabular cup. In some cases, a proximal femoral replacement was also necessary to compensate for bony defects. All patients had sufficient range of motion (ROM) at the hip with post-operative stability. It has been verified, at six and twelve months postoperatively, that there is a strong hold of the implant due to osseointegration. Additionally, in patients whose posterior acetabular wall was missing, it was discovered that the implant assisted in bone formation and covered the entire posterior surface of the implant. All patients in this study managed with this novel treatment option, proved to have a stable pelvic reconstruction with restoration of leg lengths, improvement of strength and independent ambulation at short and medium term follow-up

Previous studies have described an age-dependent distortion of bone microarchitecture for α-CGRP-deficient mice (3). In addition, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from young wildtype (WT) mice when stimulated with α-CGRP whereas loss of α-CGRP showed only little effects on bone cell metabolism of cells isolated from young α-CGRP-deficient mice. We assume that aging processes differently affect bone cell metabolism in the absence and presence of α-CGRP. To further explore this hypothesis, we investigated and compared cell metabolism of osteoblasts and bone marrow derived macrophages (BMM)/osteoclast cultures isolated from young (8–12 weeks) and old (9 month) α-CGRP-deficient mice and age matched WT controls. Isolation/differentiation of bone marrow macrophages (BMM, for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from young (8–12 weeks) and old (9 month) female α-CGRP−/− and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (crystal violet staining), proliferation (BrdU incorporation) and caspase 3/7-activity (apoptosis rate). Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and counting of multinucleated (≥ 3 nuclei), TRAP (tartrate resistant acid phosphatase) stained osteoclasts reflects osteoclast differentiation capacity. We counted reduced numbers of BMM from young α-CGRP−/− mice after initial seeding compared to young WT controls but we found no differences between old α-CGRP−/− mice and age-matched controls. Total BMM number was higher in old compared to young animals. Migration of osteoblast-like cells out of bone chips was comparable in both, young and old α-CGRP−/− and WT mice, but number of osteoblast-like cells was lower in old compared to young animals. Proliferation of old α-CGRP−/− BMM was higher when compared to age-matched WT whereas proliferation of old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation was lower. No differences in bone cell proliferation was detected between young α-CGRP−/− and age-machted WT mice. Caspase 3/7 activity of bone cells from young as well as old α-CGRP−/− mice was comparable to age-matched controls. Number of TRAP-positive multinucleated osteoclasts from young α-CGRP−/− mice was by trend higher compared to age-matched WT whereas no difference was observed in osteoclast cultures from old α-CGRP−/− mice and old WT. ALP activity, as a marker for bone formation activity, was comparable in young WT and α-CGRP−/− osteoblasts throughout all time points whereas ALP activity was strongly reduced in old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation compared to age-matched WT. Our data indicate that loss of α-CGRP results in a reduction of bone formation rate in older individuals caused by lower proliferation and reduced activity of osteogenic cells but has no profound effects on bone resorption rate. We suggest that the osteopenic bone phenotype described in aged α-CGRP-deficient mice could be due to an increase of dysfunctional matured osteoblasts during aging resulting in impaired bone formation

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

Introduction. Fully implantable systems are used commonly only after maturity. What are indications to use fully implantable systems at the femur even in children?. Materials and Methods. Implantable lengthening nails (FITBONE) were used retrograde at the femur in minimal invasive technique to correct a limb length discrepancy of >6 cm. In 5 cases a relevant deformity was corrected in the same surgery. In all cases a final step of lengthening was planned at the femur and at the tibia with fully implantable devices at maturity. Results. 18 patients with the medium age of 10,3 years (8–14) were treated. In 17 cases the goal of lengthening was achieved without any complication. In one case of proximal femoral deficiency lengthening had to be stopped because of increasing tendency of knee joint luxation. Bone formation occurred circular around the nail in all cases. Full load bearing was possible in the average after 2,2 days/mm. No technical problems occur. In one case induced deformity in the lateral plane was observed which was corrected at the final step. At the end of treatment functional and cosmetical result was perfect in all cases. Conclusions. Fully implantable motorized distraction nails are a favorable option for lengthening and deformity correction of the femur even for children older than 10 years to correct limb length discrepancy of more than 6 cm. The treatment has a low pain level, is comfortable and nearly no scars are visible

Introduction. Despite all modern advances the indications and possibilities of treatment in Legg-Calvé-Perthes Disease (LCPD) are still controversial. In the past 15 years we started using arthrodiastasis of the hip creating negative pressure over the avascular femoral head in some selected cases of LCPD in the active phasis of the disease. Now we present our experience with distraction in LCPD using the original Ilizarov circular frame for the hip. Materials and Methods. From 2015 to 2021, eight patients with LCPD were submoitted to hip distraction; all male ranging from six to 12 years (avg 8.2 years). All patients with bad prognosis, in Catterall Groups III and IV. All patients in the active phasis of the disease. Our follow-up is ranging from two to six years, with an average of 3.4 years. Results. All the preliminary results are good with recovering of range of motion of the affected hip joint in allpatients. After the first month under distraction is possible to observe new bone formation specially in the lateral pillar of the femoral head. Complications were not observed in this group of patients. Conclusions. The main point of this prospective study is to find that is possible to achieve cartilaginous growth on the femoral head stimulated by the traction offered by the external fixator, in a very short period of time. Distraction may be one method for a good treatment in patients aged six years or older with Legg-Calvé-Perthes disease in the active phase of the disease

Introduction. Revision total hip arthroplasty is often associated with acetabular bone defects. In most cases, assessment of such defects is still qualitative and biased by subjective interpretations. Three-dimensional imaging techniques and novel anatomical reconstructions using statistical shape models (SSM) allow a more impartial and quantitative assessment of acetabular bone defects [1]. The objectives of this study are to define five clinically relevant parameters and to assess 50 acetabular bone defects in a quantitative way. Methods. Anonymized CT-data of 50 hemi-pelvises with acetabular bone defects were included in the study. The assessment was based on solid models of the defect pelvis (i.e. pelvis with bone defect) and its anatomical reconstruction (i.e. native pelvis without bone defect) (Fig.1A). Five clinically relevant parameters were defined: (1) Bone loss, defined by subtracting defect pelvis from native pelvis. (2) Bone formation, defined by subtracting native pelvis from defect pelvis. Bone formation represents bone structures, which were not present in the native pelvis (e.g. caused by remodeling processes around a migrated implant). (3) Ovality, defined by the length to width ratio of an ellipse fitted in the defect acetabulum. A ratio of 1.0 would represent a circular acetabulum. (4) Lateral center-edge angle (LCE angle), defined by the angle between the most lateral edge of the cranial roof and the body Z-axis, and (5) implant migration, defined by the distance between center of rotation (CoR) of the existing implant and CoR of native pelvis (Fig. 1B). Results. All data are presented as single values as well as median and [25. th. , 75. th. ]- percentile (Fig.2). Bone loss was 53.6 [41.5, 76.7] ml with a minimum of 19.0 ml and maximum of 103.9 ml. Bone formation was 15.7 [10.5, 21.2] ml with a minimum of 3.5 ml and a maximum of 41.6 ml. Ovality was 1.3 [1.1, 1.4] with a minimum of 1.0 and a maximum of 2.0. LCE angle was 30.4° [21.5°, 40.1°] with a minimum of 11.6° and a maximum of 63.0°. Implant migration was 25.3 [15.1, 32.6] mm with a minimum of 5.4 mm and a maximum of 53.5 mm. Discussion. Within this study, 50 hemi-pelvises with acetabular bone defects were successfully quantified using five clinically relevant parameters. Application of this method provides impartial and quantitative data of acetabular bone defects, which could be beneficial in clinical practice for pre-operative planning or comparison of surgical outcomes. Including a larger number of cases, this method could even serve as a basis for a novel classification system for acetabular bone defects. For any figures or tables, please contact the authors directly

Despite the increasing availability of bone grafting materials, the regeneration of large bone defects remains a challenge. Especially infection prevention while fostering regeneration is a crucial issue. Therefore, loading of grafting material with antibiotics for direct delivery to the site of need is desired. This study evaluates the concept of local delivery using in vitro and in vivo investigations. We aim at verifying safety and reliability of a perioperative enrichment procedure of demineralized bone matrix (DBM) with gentamicin. DBM (DBMputty, DIZG, Germany) was mixed with antibiotic using a syringe with an integrated mixing propeller (Medmix Systems, Switzerland). Gentamicin, as powder or solution, was mixed with DBM at different concentrations (25 −100 mg/g DBM), release and cytotoxicity was analyzed. For in vivo analysis, sterile drill hole defects (diameter: 6 mm, depth: 15 mm) were created in diaphyseal and metaphyseal bones of sheep (Pobloth et al. 2016). Defects (6 – 8 per group and time point) were filled with DBM or DBM enriched with gentamicin (50 mg/g DBM) or left untreated. After three and nine weeks, defect regeneration was analyzed by µCT and histology. The release experiments revealed a burst release of gentamicin from DBM independent of the used amount, the sampling strategy, or the formulation (powder or solution). Gentamicin was almost completely released after three days in all set-ups. Eluates showed an antimicrobial activity against S. aureus over at least three days. Eluates had no negative effect on viability and alkaline phosphatase activity of osteoblast-like cells (partially published Bormann et al. 2014). µCT and histology of the drill hole defects revealed a reduced bone formation with gentamicin loaded DBM. After nine weeks significantly less mineralized tissue was detectable in metaphyseal defects of the gentamicin group. Histological evaluation revealed new bone formation starting at the edges of the drill holes and growing into the center over time. The amount of DBM decreased over time due to the active removal by osteoclasts while osteoblasts formed new bone. Using this mixing procedure, loading of DBM was fast, reliable and possible during surgical setting. In vitro experiments revealed a burst and almost complete release after three days, antimicrobial activity and good biocompatibility of the eluates. Gentamicin/DBM concentration was in the range of clinically used antibiotic-loaded-cement for prophylaxis and treatment in joint replacement (Jiranek et al. 2006). The delayed healing seen in vivo was unexpected due to the good biocompatibility found in vitro. A reduced healing was also seen in spinal fusion where DBM was mixed with vancomycin (Shields et al. 2017), whereas DBM with gentamicin or DBM/bioactive glass with tobramycin had no negative effect on osteoinductivity or femur defect healing, respectively (Lewis et al. 2010, Shields et al. 2016). In conclusion, loading of DBM with gentamicin showed a proper antibiotic delivery over several days, covering the critical phase shortly after surgery. Due to the faster and complete release of the antibiotic compared to antibiotic loaded cement, the amount of antibiotic should be much lower in the DBM compared to cement

Age-related fragility fractures are highly correlated with the loss of bone integrity and deteriorated morphology of the osteocytes. Previous studies have reported low-magnitude high-frequency vibration(LMHFV) promotes osteoporotic diaphyseal fracture healing to a greater extent than in age-matched normal fracture healing, yet how osteoporotic fractured bone responds to the mechanical signal has not been explored. As osteocytes are prominent for mechanosensing and initiating bone repair, we hypothesized that LMHFV could enhance fracture healing in ovariectomized metaphyseal fracture through morphological changes and mineralisation in the osteocyte Lacuno-canalicular Network(LCN). As most osteoporotic fractures occur primarily at the metaphysis, an osteoporotic metaphyseal fracture model was established. A total of 72 six-month old female Sprague-Dawley rats (n=72) were obtained(animal ethical approval ref: 16–037-MIS). Half of the rats underwent bilateral ovariectomy(OVX) and kept for 3 months for osteoporosis induction. Metaphyseal fracture on left distal femur was created by osteotomy and fixed by a plate. Rats were then randomized to (1) OVX+LMHFV(20 mins/day and 5 days/week, 35Hz, 0.3g), (2) OVX control, (3) SHAM+LMHFV, (4) SHAM control. Assessments of morphological structural changes, functional markers of the LCN(Scanning Electron Microscopy, FITC-Imaris, immunohistochemistry), mineralization status(EDX, dynamic histomorphometry) and healing outcomes(X-ray, microCT, mechanical testing) were performed at week 1, 2 and 6 post-fracture. One‐way ANOVA with post-hoc test was performed. Statistical significance was set at p < 0.05. Our results showed LMHFV could significantly enhance the morphology of the LCN. There was a 65.3% increase in dendritic branch points(p=0.03) and 93% increase in canalicular length(p=0.019) in the OVX-LMHFV group at week 2 post-fracture. Besides, a similar trend was also observed in the SHAM+LMHFV group, with a 43.4% increase in branch points and 53% increase in canaliculi length at week 2. A significant increase of E11 and DMP1 was observed in the LMHFV groups, indicating the reconstruction of the LCN. The decreasing sclerostin and increasing FGF23 at week 1 represented the active bone formation phase while the gradual increase at week 6 signified the remodelling phase. Furthermore, Ca/P ratio, mineral apposition rate and bone formation rate were all significantly enhanced in the OVX+LMHFV group. The overall bone mineral density in BV was significantly raised in the OVX+LMHFV group at week 2(p=0.043) and SHAM+LMHFV at week 6(p=0.04). Quantitative analysis of microCT showed BV/TV was significantly increased at week 2 in OVX+LMHFV group(p=0.008) and week 6(p=0.001) in both vibration groups. In addition, biomechanical testing revealed that the OVX+LMHFV group had a significantly higher ultimate load(p=0.03) and stiffness(p=0.02) at week 2. To our best knowledge, this is the first report to illustrate LMHFV could enhance osteocytes' morphology, mineralisation status and healing outcome in a new osteoporotic metaphyseal fracture animal model. Our cumulative data supports that the mechanosensitivity of bone would not impair due to osteoporosis. The revitalized osteocyte LCN and upregulated osteocytic protein markers implied a better connectivity and transduction of signals between osteocytes, which may foster the osteoporotic fracture healing process through an enhanced mineralisation process. This could stimulate further mechanistic investigations with potential translation of LMHFV to our fragility fracture patients

Aim. The aim of this work was to evaluate, via foot and ankle TC scans, the outcomes of the use of a bone substitute (CERAMENT|™G) and the growth of native bone in the treatment of osteomyelitis (OM) of the diabetic foot. Method. In nine patients from July 2014 to December 2016 we used a Calcium Sulphate Hemihydrate + Hydroxyapatite + Gentamicin Sulfate (CSH + HA + GS) compound to fill resected bone voids following surgical intervention in OM diabetic foot cases. Of these nine patients, three were female and six were male and their ages were between 49 and 72 years. Four patients had hindfoot involvement and underwent partial calcanectomy. Two patients presented a rocker-bottom Charcot foot pattern III according to Sanders and Frykberg's classification and were treated with esostectomy of the symptomatic bony prominence of the midfoot. One patient presented OM of the 3°, 4° and 5° metatarsal bones. One patient underwent partial resection of the midfoot and hindfoot with arthrodesis stabilised by an internal-external hybrid fixator. One patient with a Charcot foot pattern IV-V underwent partial talectomy and calcanectomy with arthrodesis stabilised by an internal-external hybrid fixator. In all these patients - after removal of the infected bone - we applied 10 to 20 ml CSH + HA + GS filling the residual spaces with the aim of stabilising the remaining bone fragments. The uniqueness of this product is that it induces native bone growth, while the synthetic bone disappears and antibiotic is released into the surrounding tissues. In March 2018, the above nine patients underwent foot and ankle TC scans to evaluate bone growth. Results. The first four patients showed new bone formation in the calcaneus. Two patients with previous midfoot destruction showed chaotic but stable bone formation. The patient with metatarsal OM showed partial bone healing with residual pseudoarthrosis. Both the two patients who underwent arthrodesis with hybrid fixators showed a plantigrade and stable foot even though a heel wound is still present in one of the patients. All patients except this one are now wearing suitable shoes as post-operative wounds have healed. The patient still with the heel wound is walking with an aircast brace. Conclusion. The TC scans have shown new bone formation sufficient to stabilise the foot and allow ambulation. In particular, very good results come from the filling of the calcaneus, probably due to the anatomy of the bone itself

Introduction. Hydroxyapatite and poly-L-lactide (HA/PLLA) composites are osteoconductive and biodegradable. They have already been used clinically to treat fractured bones by inducing osteosynthesis and serving as the bone filling material. During revision of total hip arthroplasty, we have grafted bone onto the bone defect and covered it with an HA/PLLA mesh instead of using a metal mesh on the non-load bearing portion of the cup (Figure 1). However, whether the interface between the HA/PLLA and the titanium alloy cup was stable remains unclear. Objectives. The purpose of this study was to determine and compare the histological osteoconductivity and osteoinductivity of HA/PLLA and titanium alloy. Methods. Semicylindrical samples (diameter: 3 mm; height: 5 mm) were fabricated from a composite unsintered-HA/PLLA containing 40% fine HA particles and from titanium alloy (Ti-6AL-4V). These two samples were combined to form one cylinder (Figure 2). Defects 3 mm in diameter were drilled into both femoral condyles of nine Japanese white rabbits, and the samples were implanted by press fitting. The rabbits were euthanized at 2, 4, 8, and 25 weeks after implantation, and undecalcified ground samples were prepared. New bone formation was examined histologically using Toluidine blue and Villanueva Goldner stains. Results. New bone formation was observed around the sample at 4 weeks, and the amount increased by 8 weeks. In addition, partial remodeling of the trabecular bones and absorption of the HA/PLLA were found at 25 weeks. Small amounts of new bone formation were found at 4 weeks between the HA/PLLA and titanium alloy materials (Figure 3: Toluidine blue stain), and the amount increased at 8 and 25 weeks. The HA/PLLA had been slightly absorbed and new bone was formed in the gap, which was close to the border between the materials, at 25 weeks. However, the amount of absorption was limited, and no new bone was found in samples where the materials were firmly in contact. Conclusions. HA/PLLA was only slight absorbed at 25 weeks, suggesting that it was stable in vivo and has good osteoconductive and osteoinductive properties. No new bone was found in the regions where the sample was stable and had no gaps between the HA/PLLA and titanium alloy, probably because there was no space for new bone to form in those regions. In contrast, new bone formation was found in gaps of more than 20 μm. Clinically, many gaps likely exist, allowing new bone formation to occur even in a stable implant. This may stabilize the HA/PLLA and titanium alloy materials for longer times. As expected, the HA/PLLA and titanium alloy were mostly stablein vivo