Introduction. Histology is still considered the gold standard method for the evaluation of soft tissues in the musculoskeletal field, thanks to the possibility of studying structures using different staining and high magnification microscopy. To overcome the intrinsic limits of this method, contrast enhanced microtomographic (CE- microCT) protocols are constantly evolving to allow 3D study of soft tissues. However, no standardized approaches are available, and many concerns exist about the alterations induced to the samples. Method. microCT/histology protocols were explored on human tendons and menisci. To enhance contrast tissues for microCT scanning 1) examethyldisilazane drying 2) 2% phosphotungstic acid (PTA) in alcoholic solution exposition and 3) 2% PTA in aqueous solution exposition were performed; to observe PTA contrast progression, three exposition and scanning times were selected. microCT images were compared to histological slices obtained from the same samples, after rehydration protocols, or from adjacent tissues portion, stained with Picrosirius red to highlight the peculiar collagenic structures. Result. Exposition times influence PTA diffusion and tissue contrast; its specificity for collagenic structure allow a clearer contrast of the tissues. Histological processing on the same samples is possible: PTA removal requires careful washing in basic solution to reduce the hardening of the sample, while drying can be reverted applying inverse protocol. Comparison with microCT images is really accurate if histology is performed on the same sample, although all protocols induce tissue shrinkage with relative packing of collagen fibers. Conclusion. The contrast approaches tested proved effective in highlighting the structures of both tendons and menisci, but the structural effects induced by tissue shrinkage do not allow a completely real microCT visualization of native tissue. Histology can be the reference method to monitor the efficacy of the contrast methods and the alterations induced to define the possibility of improvement of the technique. Acknowledgement. PR23-PAS-P4 “ADJOINT 2”- INAIL

Abstract. Objectives. To analyse the costs and benefits of sending femoral head specimens for histopathological analysis and whether our practice had changed since the original study five years ago. Methods. The cohort definition was patients who had both undergone hip hemiarthroplasties (HHAs) and had femoral head specimens sent for histopathological analysis at our tertiary care institution from 2013 to 2016. Retrospective review of clinical and electronic notes was performed on these patients for history of malignancy, histopathological diagnosis of femoral head, indication for histopathological examination and radiological studies. The total number of HHAs performed at the centre and the costs involved in analysing each femoral head specimen were identified. Results. A total of 805 HHAs were performed at the centre. We identified 56 femoral head specimens from 56 patients (6.96%) that were sent for histopathological analysis after HHA. 29 patients (51.79%) had a known history of malignancy. Three femoral head specimens (10.34%) were histologically positive for malignancy. Two patients had hip radiographs demonstrating metastasis to the femoral head. The third patient had a PET-CT scan two weeks prior to fracture that did not show signs of metastatic disease. 27 patients (48.21%) had no history of malignancy. None of their femoral head specimens were found to be histologically positive for malignancy. 10 patients had no appropriate indication for histopathological analysis. The cost savings for 27 patients with no previous history of malignancy ranged from €2,295 to €9,504. The cost savings for 10 patients with no appropriate indication ranged from €850 to €3,520. Conclusions. Histopathological analysis of femoral head specimens after HHA has little benefit for patients without a history of malignancy. The practice at our institution had not changed significantly in the past five years. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Objectives. This study aimed to evaluate the histological and mechanical features of tendon healing in a rabbit model with second-harmonic-generation (SHG) imaging and tensile testing. Materials and Methods. A total of eight male Japanese white rabbits were used for this study. The flexor digitorum tendons in their right leg were sharply transected, and then were repaired by intratendinous stitching. At four weeks post-operatively, the rabbits were killed and the flexor digitorum tendons in both right and left legs were excised and used as specimens for tendon healing (n = 8) and control (n = 8), respectively. Each specimen was examined by SHG imaging, followed by tensile testing, and the results of the two testing modalities were assessed for correlation. Results. While the SHG light intensity of the healing tendon samples was significantly lower than that of the uninjured tendon samples, 2D Fourier transform SHG images showed a clear difference in collagen fibre structure between the uninjured and the healing samples, and among the healing samples. The mean intensity of the SHG image showed a moderate correlation (R. 2. = 0.37) with Young’s modulus obtained from the tensile testing. Conclusion. Our results indicate that SHG microscopy may be a potential indicator of tendon healing. Cite this article: E. Hase, K. Sato, D. Yonekura, T. Minamikawa, M. Takahashi, T. Yasui. Evaluation of the histological and mechanical features of tendon healing in a rabbit model with the use of second-harmonic-generation imaging and tensile testing. Bone Joint Res 2016;5:577–585. DOI: 10.1302/2046-3758.511.BJR-2016-0162.R1

Summary Statement. Atypical femoral fractures consist of a thin fracture line extending through the lateral cortex. The adjacent bone is undergoing resorption and mechanical abrasion and is often replaced with woven bone. The mechanical environment seems to inhibit healing. Background. The pathophysiology behind bisphosphonate-associated atypical femoral fractures remains unclear. Histological findings at the fracture site itself might provide important clues. So far only one case describing the histological appearance of the fracture has been published. Methods. Between 2008 and 2013, bone biopsies comprising the fracture site were collected from 8 patients with 4 displaced and 4 undisplaced atypical femoral fractures. Seven female patients reported continuous bisphosphonate use for an average of 9.5 years. One patient was a man, not using bisphosphonates. The bone biopsies were evaluated histologically, with Fourier transformed infrared imaging (FTIR) and micro-computed tomography. Results. The 4 undisplaced fractures engaged the whole cortical thickness and comprised a 150 to 200 µm wide, meandering fracture gap filled with amorphous necrotic material. Von Kossa staining showed occasional mineralised elements with bony structure within the amorphic material. Active resorption and remodeling was common in the close vicinity of the fracture, but seldom reached into the fracture gap. In some areas, the bone adjacent to the gap appeared to undergo fragmentation and disintegration, possibly due to abrasion. Woven bone was common adjacent to the fracture gap, and appeared to have been formed in defects caused by abrasion or where resorption cavities had reached into the fracture gap. Periosteal and endosteal callus was found in all cases. Far away from the fracture, large areas of osteonal bone with only empty osteocyte lacunae were found in some samples. In one patient, the remodeling process bridged the fracture gap at some points. The fracture was otherwise similar to the other undisplaced fractures. This patient had suffered from thigh pain since her bisphosphonate treatment was discontinued 18 months earlier, when the atypical fracture was diagnosed. Discussion. Atypical femoral fractures show signs of increased remodeling in the vicinity of the fracture gap. The narrow width of the gap and its necrotic contents suggest that micromotion leads to strains between the fracture fragments that precludes survival of ingrowing cells. Moreover, there seemed to be continuous mechanical fragmentation of the bone at the crack, and replacement of fragmented areas with woven bone. Thus, it appears that the fracture line is not static, but moves in the bone over time, like the changes in the course of a meandering river

Acetabular dysplasia was produced in 24 immature white rabbits. A rotational acetabular osteotomy was then carried out and radiological and histological studies of the articular cartilage were made. In the hips which did not undergo osteotomy, radiographs at 26 weeks showed that residual subluxation remained and arthritic changes such as narrowing of the joint space or dislocation were still seen. However, in the operated group there was a remarkable increase in cover, but arthritic changes were not observed. After 24 weeks, the Mankin grading score in the operated group was significantly lower than that in the non-operated group. The latter hips showed an irregular surface of the cartilage, exfoliation and proliferation of synovial tissue. In those undergoing osteotomy, primary cloning of chondrocytes or hypercellularity was seen and at 24 weeks after operation and metaplasia of the cartilage in the fibrous tissue was observed in the boundary between the medial area of the acetabulum and the acetabular fossa

Digital Ventilated Cages (DVC) offer an innovative technology to obtain accurate movement data from a single mouse over time [1]. Thus, they could be used to determine the occurrence of a tendon damage event as well as inform on tissue regeneration [2,3]. Therefore, using the mouse model of tendon experimental damage, in this study it has been tested whether the recovery of tissue microarchitecture and of extracellular matrix (ECM) correlates with the motion data collected through this technology. Mice models were used to induce acute injury in Achilles tendons (ATs), while healthy ones were used as control. During the healing process, the mice were housed in DVC cages (Tecniplast) to monitor animal welfare and to study biomechanics assessing movement activity, an indicator of the recovery of tendon tissue functionality. After 28 days, the AT were harvested and assessed for their histological and immunohistochemical properties to obtain a total histological score (TSH) that was then correlated to the movement data. DVC cages showed the capacity to distinguish activity patterns in groups from the two different conditions. The data collected showed that the mice with access to the mouse wheel had a higher activity as compared to the blocked wheel group, which suggests that the extra movement during tendon healing improved motion ability. The histological results showed a clear difference between different analyzed groups. The bilateral free wheel group showed the best histological recovery, offering the highest TSH score, thus confirming the results of the DVC cages and the correlation between movement activity and structural recovery. Data obtained showed a correlation between TSH and the DVC cages, displaying structural and movement differences between the tested groups. This successful correlation allows the usage of DVC type cages as a non-invasive method to predict tissue regeneration and recovery. Acknowledgements: This research is part of the P4FIT project ESR13, funded by the H2020-ITN-EJD MSCA grant agreement No.955685

Obesity is associated with poor outcomes and increased risk of failure after rotator cuff (RC) repair surgery. The effect of diet-induced obesity (DIO) on enthesis healing has not been well characterised and whether its effects can be reversed with dietary intervention is unknown. We hypothesised that DIO would result in inferior enthesis healing in a rat model of RC repair and that dietary intervention in the peri-operative period would improve enthesis healing. A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were cullers and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively. DIO was established in the HFD and HF-CD groups prior to surgery, and subsequently reversed in the HF-CD group after surgery. At 12 weeks post-surgery, plasma leptin concentrations were higher in the HFD group compared to the CD group (5.28 vs. 2.91ng/ml, P=0.003). Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD compared to the CD group at 12 weeks (overall histological score 6.20 (P=0.008), 4.98 (P=0.001) and 8.68 out of 15, respectively). The repaired entheses in the HF-CD group had significantly lower (26.4 N, P=0.028) load-at-failure 12 weeks post-surgery compared to the CD group (34.4 N); while the HFD group was low, but not significantly different (28.1 N, P=0.096). Body mass at the time of surgery, plasma leptin and body fat percentage were negatively correlated with histological scores and plasma leptin with load-at-failure 12 weeks post-surgery. DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Exploring interventions that improve the metabolic state of obese patients and counselling patients appropriately about their modest expectations after repair should be considered

Introduction. The most frequent diagnosis in young adults undergoing total hip arthroplasty (THA) is osteonecrosis of the femoral head (ONFH), an evolving and disabling condition with an increasing prevalence worldwide. Treatment of ONFH remains a challenge mainly because of a lack of understanding of the disease's pathophysiological basis. This study investigated the biological processes that could be affected by ONFH by comparing the microstructure, histological characteristics and transcriptomic profile of trabecular bone from the femoral head (FH) and the intertrochanteric region (IT) of patients suffering from this condition. Method. A total of 18 patients with idiopathic ONFH undergoing THA in our institution were included. Trabecular bone explants were taken intraoperatively from the FH and the IT of patients. Bone microstructure was examined by micro-computed tomography (micro-CT). After bone sectioning, histological features were studied by hematoxylin and eosin staining. Differential gene expression was investigated using a microarray platform. Result. Micro-CT imaging showed higher trabecular separation and lower trabecular thickness and bone volume in trabecular bone from the FH than from the IT. Histological staining revealed that the number of osteoblasts on the bone surface and the percentage of empty lacunae were higher in trabecular bone from the FH. Transcriptome analysis identified a differential signature in trabecular bone from the FH compared to the IT. The gene ontology analyses of the genes overexpressed in trabecular bone from the FH revealed a range of enriched biological processes related to cell division and immune response. In contrast, most downregulated transcripts were involved in bone formation. Conclusion. This study identified changes in the microarchitecture, histological features and transcriptomic signature of trabecular bone from the FH of patients with idiopathic ONFH, which might underlie the pathophysiology of this condition. This work was supported by PI22/00939 grant from ISCIII-FEDER-MICINN-AES and Luis Alvarez grant from IdiPAZ

Introduction. The Achilles tendon is the thickest and strongest tendon in the human body. Even though the tendon is so strong, it is one of the most frequently injured tendons. Treatment of patients after rupture is planned conservatively and surgically. Conservative treatment is generally applied to elderly patients with sedentary lives. If the treatment is surgical, it can be planned as open surgery or percutaneous surgery. In our study with rabbits, we wrapped a membrane made of plga (polylactic-co-glycolic acid) nanotubes impregnated with type 1 collagen around the tendon in rabbits that underwent open Achilles tendon repair surgery. After surgery, biomechanical and histological tests were performed on the tendons. Method. In the study consisting of 24 rabbits, 2 groups were created by random distribution. In the study group, after the Achilles tendon rupture was created, a type 1 collagen-impregnated plga-based membrane was placed around the tendon after the repair of 1 modified Kesslerr suture. In the control group, after the Achilles tendon rupture was created, 1 modified Kessler suture and Tendon repair was performed with the application of 3 primary sutures. At the end of the 6th week of the study, the rabbits in 2 groups were randomly distributed and histological examination was performed. Additionally, biomechanical testing was performed. Bonar and Movın scoring were used in histological examinations. Result. As a result of biomechanical tests, it was seen that the resistance of the tendon against rupture was higher in the study group than in the control group. In addition, it was observed that the tendon rupture time was longer in the study group than in the control group. Histological examinations gave supportive results from biomechanical tests. Conclusion. We think that the use of collagen-impregnated plga-based nanotubes in the surgical treatment of Achilles tendon ruptures has a positive healing effect. Although we think that the return to normal life after surgery may be faster, we believe that more clinical studies are needed

Surgeons treating fractures with many small osteochondral fragments have often expressed the clinical need for an adhesive to join such fragments, as an adjunct to standard implants. If an adhesive would maintain alignment of the articular surfaces and subsequently heal it could result in improved clinical outcomes. However, there are no bone adhesives available for clinical indications and few pre-clinical models to assess safety and efficacy of adhesive biomaterial candidates. A bone adhesive candidate based on water, α-TCP and an amino acid phosphoserine was evaluated in-vivo in a novel murine bone core model (preliminary results presented EORS 2019) in which excised bone cores were glued back in place and harvested @ 0, 3, 7, 14, 28 and 42days. Adhesive pull-out strength was demonstrated 0–28 days, with a dip at 14 days increasing to 11.3N maximum. Histology 0–42 days showed the adhesive progressively remodelling to bone in both cancellous and cortical compartments with no signs of either undesirable inflammation or peripheral ectopic bone formation. These favourable results suggested translation to a large animal model. A porcine dental extraction socket model was subsequently developed where dental implants were affixed only with the adhesive. Biomechanical data was collected @ 1, 14, 28 and 56 days, and histology at 1,14,28 and 56 days. Adhesive strength assessed by implant pull-out force increased out to 28 days and maintained out to 56 days (282N maximum) with failure only occurring at the adhesive bone interface. Histology confirmed the adhesive's biocompatibility and osteoconductive behavior. Additionally, remodelling was demonstrated at the adhesive-bone interface with resorption by osteoclast-like cells and followed by new bone apposition and substitution by bone. Whilst the in-vivo dental implant data is encouraging, a large animal preclinical model is needed (under development) to confirm the adhesive is capable of healing, for example, loaded osteochondral bone fragments. Acknowledgements: The murine study was supported, in part, by the Swedish Foundation for Strategic Research (#RMA15-0110)

Introduction. The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels. Method. Chemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques. Result. A 3D scaffold using ELR that combines various peptides with different degradation rates to guide both angiogenesis and neurogenesis has been developed. Notably, scaffolds with different degradation rates promoted distinct patterns of vascularization and innervation, facilitating integration with host tissue. This work demonstrates the potential for tailored tissue engineering, where the scaffold's bioactivities and degradation rates can control angiogenesis and neurogenesis. In an animal model of medication-related osteonecrosis of the jaw (MRONJ), the scaffold showed promising results in promoting bone regeneration in a necrotic environment, as confirmed by histological and imaging analyses. This study opens avenues for novel tissue-engineering strategies where precise control over vascularization and nerve growth is crucial. Conclusion. A groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold achieves effective time/space control over necrotic bone regeneration. The authors are grateful for funding from the Spanish Government (PID2020-118669RA-I00)

The primary aim of this study was to compare the clinical outcomes of osteoid osteoma (OO) between the group of patients with the presence of nidus on biopsy samples from radiofrequency ablation (RFA) with those without nidus. Secondly, we aimed to examine other factors that may affect the outcomes of OO reflecting our experience as a tertiary orthopaedic oncology centre. We retrospectively reviewed 88 consecutive patients diagnosed with OO treated with RFA between November 2005 and March 2015, consisting of 63 males (72%) and 25 females (28%). Sixty-six patients (75%) had nidus present in their biopsy samples. Patients’ mean age was 17.6 years (4-53). Median duration of follow-up was 12.5 months (6-20.8). Lesions were located in the appendicular skeleton in seventy-nine patients (90%) while nine patients (10%) had an OO in the axial skeleton. Outcomes assessed were based on patients’ pain alleviation (partial, complete, or no pain improvement) and the need for further interventions. Pain improvement in the patient group with nidus in histology sample was significantly better than the group without nidus (OR 7.4, CI 1.35-41.4, p=0.021). The patient group with nidus on biopsy demonstrated less likelihood of having a repeat procedure compared to the group without nidus (OR 0.092, CI 0.016-0.542, p=0.008). Our study showed significantly better outcomes in pain improvement in appendicular lesions compared to the axially located lesions (p = 0.005). Patients with spinal lesions tend to have relatively poor pain relief than those with appendicular or pelvic lesions (p=0.007). Patients with nidus on histology had better pain alleviation compared to patients without nidus. The histological presence of nidus significantly reduces the chance of repeat interventions. The pain alleviation of OO following RFA is better in patients with appendicular lesions than spinal or axially located lesions

To evaluate the therapeutic effect of Pulsed Electromagnetic Field (PEMF) in the treatment of meniscal tears in the avascular region. Seventy-two twelve-week-old male Sprague-Dawley rats with full-thickness longitudinal medial meniscal tears in the avascular region were divided into 3 groups: control group (G. con. ), treated with classic signal PEMF (G. classic. ), and high slew rate signal PEMF(G. HSR. ). The HSR signal has the same pulse and burst frequencies as the classic signal, but with a higher slew rate. Macroscopic observation and histological analysis of the meniscus and articular cartilage were performed to evaluate the meniscal healing and progressions of osteoarthritis. The synovium was harvested for histological and immunofluorescent analysis to assess the intra-articular inflammation. The meniscal healing, articular cartilage degeneration, and synovitis were quantitatively evaluated according to their respective scoring system. Dramatic degenerative changes of the meniscus and articular cartilage were noticed during gross observation and histological evaluation in the control group at 8 weeks. However, the menisci in the two treatment groups were restored to normal morphology with a smooth surface and shiny white color. Particularly, the HSR signal remarkably enhanced the fibrochondrogenesis and accelerated the remodeling process of the regenerated tissue. The meniscal healing scores of PEMF treatment groups were significantly higher than those in the control group at 8 weeks. Specifically, the HSR signal showed a significantly higher meniscal repair score than the classic signal at week 8 (P < .01). The degeneration score (G. con. versus G. classic. : P < .0001; Gcon versus G. HSR. : P < .0001) and synovitis score (G. con. versus Gclassic: P < .0001; G. con. versus G. HSR. : P = .0002) of the control groups were significantly higher than those in the two treatment groups. PEMF promoted the healing of meniscal tears in the avascular region and restored the injured meniscus to its structural integrity in a rat model. Compared to the classic signal, the HSR signal showed the increased capability to promote fibrocartilaginous tissue formation and modulate the inflammatory environment and therefore protected the knee joint from post-traumatic osteoarthritis development

To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model. 5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10. 6. and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls. All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings. The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested

In chronically infected fracture non-unions, treatment requires extensive debridement to remove necrotic and infected bone, often resulting in large defects requiring elaborate and prolonged bone reconstruction. One approach includes the induced membrane technique (IMT), although the differences in outcome between infected and non-infectious aetiologies remain unclear. Here we present a new rabbit humerus model for IMT secondary to infection, and, furthermore, we compare bone healing in rabbits with a chronically infected non-union compared to non-infected equivalents. A 5 mm defect was created in the humerus and filled with a polymethylmethacrylate (PMMA) spacer or left empty (n=6 per group). After 3 weeks, the PMMA spacer was replaced with a beta-tricalcium phosphate (chronOs, Synthes) scaffold, which was placed within the induced membrane and observed for a further 10 weeks. The same protocol was followed for the infected group, except that four week prior to treatment, the wound was inoculated with Staphylococcus aureus (4×10. 6. CFU/animal) and the PMMA spacer was loaded with gentamicin, and systemic therapy was applied for 4 weeks prior to chronOs application. All the animals from the infected group were culture positive during the first revision surgery (mean 3×10. 5. CFU/animal, n= 12), while at the second revision, after antibiotic therapy, all the animals were culture negative. The differences in bone healing between the non-infected and infected groups were evaluated by radiography and histology. The initially infected animals showed impaired bone healing at euthanasia, and some remnants of bacteria in histology. The non-infected animals reached bone bridging in both empty and chronOs conditions. We developed a preclinical in vivo model to investigate how bacterial infection influence bone healing in large defects with the future aim to explore new treatment concepts of infected non-union

Successful application of patient derived cells to engineer vascularized bone grafts is often hampered by low cell numbers and lengthy in vitro expansion. With sound induced morphogenesis (SIM), local cell density enhancement was shown to improve microvasculature formation at lower cell concentration than conventional methods [1]. SIM takes advantage of hydrodynamic forces that act on cells to arrange them within a hydrogel. Following, we are evaluating the potential of cell-hydrogel biografts with high local cell density to improve the therapeutic efficacy in clinical scenarios such as anastomosis or bone formation within non-union fractures. To assess anastomosis, human umbilical vein endothelial cells (HUVEC) and human mesenchymal stromal cells (MSC) were mixed at a 1:1 ratio in PEG-based or Dextran-based hydrogels at a final concentration of 2×10. 6. cells×mL. -1. For ectopic bone formation, MSC were resuspended in PEG-based hydrogels at 2×10. 6. or 5×10. 6. cells×mL. -1. , with or without BMP-2. Cells were assembled into distinct patterns at a frequency of 60 Hz. Four biografts of 4 × 9 mm. 2. were implanted at the back of nude mice (total of 7 animals) and harvested after 2 or 8 weeks. Explants were fixed and imaged as whole constructs or embedded in paraffin for histological analysis. Upon explantation, microscopic evaluation indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks and formed a pre-vascular network. In the second study, ectopic bone formation was more pronounced in areas of higher local cell density based on visual inspection. Ongoing experiments are further characterizing bone formation by micro-CT and histological evaluation. Our results indicate that local cell density enhancement by sound requires a lower initial cell concentration than conventional, static seeding methods to achieve comparable microvasculature structures or local osteogenesis

Treatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remain a challenge. A novel surgical technique named Tibial Cortex Transverse Transport has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In present study, we aimed to explore the wound healing effects after undergoing this novel technique via multiple ways. A novel rat model of Tibial Cortex Transverse Transport was established with a designed external fixator and effects on wound healing were investigated. All rats were randomized into 3 groups, with 12 rats per group: sham group (negative control), fixator group (positive control) and Tibial Cortex Transverse Transport group. Laser speckle perfusion imaging, vessel perfusion, histology and immunohistochemistry were used to evaluate the wound healing processes. Gross and histological examinations showed that Tibial Cortex Transverse Transport technique accelerated wound closure and enhanced the quality of the newly formed skin tissues. In Tibial Cortex Transverse Transport group, HE staining demonstrated a better epidermis and dermis recovery, while immune-histochemical staining showed that Tibial Cortex Transverse Transport technique promoted local collagen deposition. Tibial Cortex Transverse Transport technique also benefited to angiogenesis and immunomodulation. In Tibial Cortex Transverse Transport group, blood flow in the wound area was higher than that ofother groups according to laser speckle imaging with more blood vessels observed. Enhanced neovascularization was seen in the Tibial Cortex Transverse Transport group with double immune-labelling of CD31 and α-SMA. The M2 macrophages at the wound site in the Tibial Cortex Transverse Transport group was also increased. Tibial cortex transverse transport technique accelerated wound healing through enhanced angiogenesis and immunomodulation

Glutamate regulates the expression of apoptosis-related genes and triggers the apoptosis of fibroblasts in rotator cuff tendons. Subacromial bursitis is always accompanied by symptomatic rotator cuff tear (RCT). However, no study has been reported on the presence of glutamate in subacromial bursa and on its involvement of shoulder pain in patients who had RCT. The purposes of this study were to determine whether the glutamate expression in subacromial bursa is associated with the presence of RCT and with the severity of shoulder pain accompanying RCT. Subacromial bursal tissues were harvested from patients who underwent arthroscopic rotator cuff tendon repair or glenoid labral repair with intact rotator cuff tendon. Glutamate tissue concentrations were measured, using a glutamate assay kit. Expressions of glutamate and its receptors in subacromial bursae were histologically determined. The sizes of RCT were determined by arthroscopic findings, using the DeOrio and Cofield classification. The severity of shoulder pain was determined, using visual analog scale (VAS). Any associations between glutamate concentrations and the size of RCT were evaluated, using logistic regression analysis. The correlation between glutamate concentrations and the severity of pain was determined, using the Pearson correlation coefficient. Differences with a probability <0.05 were considered statistically significant. Glutamate concentrations showed significant differences between the torn tendon group and the intact tendon group (P = 0.009). Concentrations of glutamate significantly increased according to increases in tear size (P < 0.001). In histological studies, the expressions of glutamate and of its ionotropic and metabotropic receptors have been confirmed in subacromial bursa. Glutamate concentrations were significantly correlated with pain on VAS (Rho=0.56 and P =0.01). The expression of glutamate in subacromial bursa is significantly associated with the presence of RCT and significantly correlated with its accompanying shoulder pain. Acknowledgements: This research was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A3A01018955 and 2017R1D1A1B03035232)

Meniscal injuries affect over 1.5 million people across Europe and the USA annually. Injury greatly reduces knee joint mobility and quality of life and frequently leads to the development of osteoarthritis. Tissue engineered strategies have emerged in response to a lack of viable treatments for meniscal pathologies. However, to date, constructs mimicking the structural and functional organisation of native tissue, whilst promoting deposition of new extracellular matrix, remains a bottleneck in meniscal repair. 3D bioprinting allows for deposition and patterning of biological materials with high spatial resolution. This project aims to develop a biomimetic 3D bioprinted meniscal substitute. Meniscal tissue was characterised to effectively inform the design of biomaterials for bioprinting constructs with appropriate structural and functional properties. Histology, gene expression and mass spectrometry were performed on native tissue to investigate tissue architecture, matrix components, cell populations and protein expression regionally across the meniscus. 3D laser scanning and magnetic resonance imaging were employed to acquire the external geometrical information prior to fabrication of a 3D printed meniscus. Bioink suitability was investigated through regional meniscal cell encapsulation in blended hydrogels, with the incorporation of growth factors and assessed for their suitability through rheology, scanning electron microscopy, histology and gene expression analysis. Meniscal tissue characterisation revealed regional variations in matrix compositions, cellular populations and protein expression. The process of imaging through to 3D printing highlighted the capability of producing a construct that accurately replicated meniscal geometries. Regional meniscal cell encapsulation into hydrogels revealed a recovery in cell phenotype, with the incorporation of growth factors into the bioink's stimulating cellular re-differentiation and improved zonal functionality. Meniscus biofabrication highlights the potential to print patient specific, customisable meniscal implants. Achieving zonally distinct variations in cell and matrix deposition highlights the ability to fabricate a highly complex tissue engineered construct. Acknowledgements: This work was undertaken as part of the UK Research and Innovation (UKRI)-funded CDT in Advanced Biomedical Materials

Although bone morphogenetic protein 2 (BMP-2) has been FDA-approved for spinal fusion for decades, its disadvantages of promoting osteoclast-based bone resorption and suboptimal carrier (absorbable collagen sponge) leading to premature release of the protein limit its clinical applications. Our recent study showed an excellent effect on bone regeneration when BMP-2 and zoledronic acid (ZA) were co-delivered based on a calcium sulphate/hydroxyapatite (CaS/HA) scaffold in a rat critical-size femoral defect model. Therefore, the aim of this study was to evaluate whether local application of BMP-2 and ZA released from a CaS/HA scaffold is favorable for spinal fusion. We hypothesized that CaS/HA mediated controlled co-delivery of rhBMP-2 and ZA could show an improved effect in spinal fusion over BMP-2 alone. 120, 8-week-old male Wistar rats (protocol no. 25-5131/474/38) were randomly divided into six groups in this study (CaS/HA, CaS/HA + BMP-2, CaS/HA + systemic ZA, CaS/HA + local ZA, CaS/HA + BMP-2 + systemic ZA, CaS/HA + BMP-2 + local ZA). A posterolateral spinal fusion at L4 to L5 was performed bilaterally by implanting group-dependent scaffolds. At 3 weeks and 6 weeks, 10 animals per group were euthanized for µCT, histological staining, or mechanical testing. µCT and histological results showed that the CaS/HA + BMP-2 + local ZA group significantly promoted bone regeneration than other treated groups. Biomechanical testing showed breaking force in CaS/HA + BMP + local ZA group was significantly higher than other groups at 6 weeks. In conclusion, the CaS/HA-based biomaterial functionalized with bioactive molecules rhBMP-2 and ZA enhanced bone formation and concomitant spinal fusion outcome. Acknowledgements: Many thanks to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology)