Superior team performance in surgery leads to fewer technical errors, reduced mortality, and improved patient outcomes. Scrub nurses are a pivotal part of this team, however they have very little structured training, leading to high levels of stress, low confidence, inefficiency, and potential for harm. Immersive virtual reality (iVR) simulation has demonstrated excellent efficacy in training surgeons. We tested the efficacy of an iVR curriculum for training scrub nurses in performing their role in an anterior approach total hip arthroplasty (AA-THA).

Sixty nursing students were included in this study and randomised in a 1:1 ratio to learning the scrub nurse role for an AA-THA using either conventional training or iVR. The training was derived through expert consensus with senior surgeons, scrub nurses and industry reps. Conventional training consisted of a 1-hour seminar and 2 hours of e-learning where participants were taught the equipment and sequence of steps. The iVR training involved 3 separate hour-long sessions where participants performed the scrub nurse role with an avatar surgeon in a virtual operation. The primary outcome was their performance in a physical world practical objective assessment with real equipment. Data were confirmed parametric using the Shapiro-Wilk test and means compared using the independent samples student's t-test.

53 participants successfully completed the study (26 iVR, 27 conventional) with a mean age of 31±9 years. There were no significant differences in baseline characteristics or baseline knowledge test scores between the two groups (p>0.05). The iVR group significantly outperformed the conventionally trained group in the real-world assessment, scoring 66.9±17.9% vs 41.3±16.7%, p<0.0001.

iVR is an easily accessible, low cost training modality which could be integrated into scrub nursing curricula to address the current shortfall in training. Prolonged operating times are strongly associated with an increased risk of developing serious complications. By upskilling scrub nurses, operations may proceed more efficiently which in turn may improve patient safety.

Ligament integrity is directly associated with ankle stability. Nearly 40% of ankle sprains result in chronic ankle instability, affecting biomechanics and potentially causing osteoarthritis. Ligament replacement could restore stability and avoid this degenerative pathway, but a greater understanding of ankle ligament behaviour is required. Additionally, autograft or allograft use is limited by donor-site morbidity and inflammatory responses respectively. Decellularised porcine grafts could address this, by removing cellular material to prevent acute immune responses, while preserving mechanical properties.

This project will characterise commonly injured ankle ligaments and damage mechanisms, identify ligament reconstruction requirements, and investigate the potential of decellularised porcine grafts as a replacement material.

Several porcine tendons were evaluated to identify suitable candidates for decellularisation. The viscoelastic properties of native tissues were assessed using dynamic mechanical analysis (DMA), followed by ramp to ‘sub-rupture’ at 1% strain/s, and further DMA. Multiple samples (n=5) were taken along the graft to assess variation along the tendon.

When identifying suitable porcine tendons, a lack of literature on human ankle ligaments was identified. Inconsistencies in measurement methods and properties reported makes comparison between studies difficult.

Preliminary testing on porcine tendons suggested there is little variation in viscoelastic properties along the length of tendon. Testing also suggested strain rates of 1%/s sub-rupture was not large enough to affect viscoelastic properties (no changes in storage or loss moduli or tanẟ). Further testing is underway to improve upon low initial sample numbers and confirm these results, with varying strain rates to identify suitable sub-rupture sprain conditions.

This work highlights need for new data on human ankle ligaments to address knowledge gaps and identify suitable replacement materials. Future work will generate this data and decellularise porcine tendons of similar dimensions. Collagen damage will be investigated using histology and lightsheet microscopy, and viscoelastic changes through DMA.

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.

To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.

pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 & E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p<0.05).

Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.

Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

Abstract

Abstract

The concept of decellularised xenografts as a basis for anterior cruciate ligament (ACL) reconstruction was introduced to overcome limitations in alternative graft sources such as substantial remodelling delaying recovery and donor site morbidity. This study aimed to measure the biomechanical properties of decellularised porcine super flexor tendon (pSFT) processed to create ACL grafts of varying diameters, with a view to facilitating production of stratified ‘off the shelf’ products with specified functional properties for use in ACL reconstructive surgery.

Decellularisation was carried out using a previously established procedure, including antibiotic washes, low concentration detergent (0.1% sodium dodecyl sulphate) washes and nuclease treatments. Decellularised pSFTs were prepared to create double-bundle grafts of 7, 8 and 9mm diameter (n=6 in each group). Femoral and tibial fixations were simulated utilising Arthrex suspension devices (Tightrope®) and interference screws in bovine bone respectively.

Dynamic stiffness and creep were measured under cyclic loading between 50–250N for 1000 cycles at 1Hz. This was followed by ramp to failure at 200mm/min from which linear stiffness and load at failure were measured. Data were analysed using either 1- or 2-way ANOVA as appropriate with Tukey post-hoc analysis (p<0.05).

Significant differences were found between all groups for dynamic stiffness and between 7 & 9mm and 8 & 9mm groups for dynamic creep. Significant differences were also found between 7, 8 & 9mm groups for linear stiffness (167.8±4.9, 186.9±16.6 & 216.3±12.4N/mm respectively), but no significant differences were found between groups for load at failure (531.5±58.9, 604.1±183.3 & 627.9±72.4N respectively).

This study demonstrated that decellularised pSFTs possess comparable biomechanical properties to other ACL graft options (autografts and allografts). Furthermore, grafts can be stratified by their diameter to provide varying biomechanical profiles depending on the anatomy and individual needs of the recipient.

We have developed a decellularised porcine superflexor tendon (pSFT), which has shown promising regenerative capacity in an ovine model of anterior cruciate ligament (ACL) repair. This study investigated the strain rate dependent and dynamic mechanical properties of native and decellularised pSFTs.

Decellularisation was carried out using a previously established procedure, including antibiotic washes, low concentration detergent (0.1% sodium dodecyl sulphate) washes and nuclease treatments.

Three different strain rates were employed: 1, 10 & 100%s-1 (n=6 for all groups). Toe-region modulus (E0), linear-region modulus (E1), transition coordinates (εT, σT), tensile strength (UTS) and failure strain were calculated. For DMA, specimens were loaded between 1 & 5MPa with increasing frequency up to 2Hz. Dynamic (E*), storage (E') and loss (E'') moduli, and tan delta were calculated for native and decellularised groups (n=6). Data was analysed by 2-way ANOVA and Tukey post-hoc test (p<0.05).

For decellularised tendons, altering the strain rate did not affect any of the static tensile properties. For native pSFTs, the UTS, failure strain and E1 were not affected by changing the strain rate. Increasing the strain rate significantly increased E0 (1% vs 10% and 1% vs 100%) and σT (1% vs 100%) and decreased εT (1% vs 10% and 1% vs 100%) for native pSFT. E*, E' and E'' were all significantly reduced in decellularised specimens compared to native controls across all frequencies investigated. No significant differences were found for tan delta.

Evidence of strain rate dependency was witnessed in the native pSFTs by increase of the toe region modulus and displacements of the transition point coordinates. This response was not seen in the tissue following decellularisation. DMA demonstrated a reduction in dynamic, storage and loss moduli. Tan delta (E''/E') remained unchanged, indicating reductions in solid and fluid components are interlinked.

Acellular porcine super flexor tendon (pSFT) offers a promising solution to replacement of damaged anterior cruciate ligament [1]. It is desirable to package and terminally sterilise the acellular grafts to eliminate any possible harmful pathogens. However, irradiation techniques can damage the collagen ultra-structure and consequently reduce the mechanical properties [2]. The aims of this study were to investigate the effects of irradiation sterilisation of varying dosages on the biomechanical properties of the acellular pSFT.

Tendons were decellularised using a previously established protocol [1] and subjected to irradiation sterilisation using either 30 kGy gamma, 55 kGy gamma, 34 kGy E-beam, 15 kGy gamma, 15 kGy E-beam and (15+15) kGy E-beam (fractionated dose). Specimens then underwent stress relaxation and strength testing at 0 and 12 months post sterilisation to determine whether any effect on these properties was progressive. For stress relaxation testing, specimens were analysed using a Maxwell-Wiechert model. For strength testing, the ultimate tensile strength, Young's modulus and failure strain were assessed.

Significant differences were found which demonstrated that all irradiation treatments had an effect on the time-independent and time-dependent viscoelastic properties of irradiated tendons compared to per-acetic acid only treated controls. Interestingly, no significant differences were found between the irradiated groups. Similar trends were found for the strength testing properties. No significant differences were found between groups at 0 and 12 months.

Tendons retained sufficient biomechanical properties following sterilisation, however it was notable that there were no significant differences between the irradiated groups, as it was believed higher dosages would lead to a greater reduction in the mechanical properties. The changes observed were not altered further after 12 months storage, indicating the acellular pSFT graft has a stable shelf-life.

Purpose: Bone destruction occurs due to the growth of primary malignant bone tumours (sarcomas) that are often not amendable to surgery. Bone resorption is carried out by osteoclasts which are formed from cells of the mononuclear phagocyte system. Primary malignant bone tumours contain tumour-associated macrophages (TAMs) in addition to neoplastic cells. The aim of the study was to determine the cellular and humoral conditions required for TAM-osteoclast differentiation and to assess the affect of an anti-osteolytic agent on osteoclastic bone resorption.

Methods: TAMs were isolated form bone and soft tissue sarcoma by collagenase digestion and cultured in the presence of RANKL and M-CSF on coverslips and dentine slices for up to 21 days. The extent of osteoclast formation and resorption was determined by expression of osteoclast markers (TRAP, VNR, cathepsin K) in cell cultures on coverslips and the extent of lacunar resorption in cell cultures on dentine slices.

Results: Osteoclast formation occurred only when RANKL and M-CSF were added to the TAM cultures. This resulted in the formation of numerous mononuclear multinucleated cells which were strongly TRAP, VNR and cathepsin K positive. In cell cultures on dentine slices, it was noted that these cells were capable of extensive lacunar resorption with formation of multiple large lacunar resorption pits. The addition of the bisphosphonate zoledronate to the cell cultures resulted in inhibition of osteoclast formation and complete absence of lacunar resorption.

Conclusion: These findings indicate that sarcoma-associated macrophages are capable of differentiating into osteoclasts and that both RANKL and M-CSF are required for this to occur. This process is likely to contribute to tumour osteolysis associated with the growth of sarcomas in bone. Further assessment of the use of inhibitors of osteoclast formation/resorption, is also indicated by our results.

Mast cells (MC), the tissue-based effector cells in allergic diseases, have many functions. Within bone tissue, they have been linked with new blood vessel formation and marrow fibrosis and it has been proposed that they are capable of promoting osteoclastic bone resorption. MC numbers are known to increase in a number of osteolytic conditions e.g. osteoporosis, hyperparathyroidism and periodontitis. In fracture callus, too, large numbers of MC are present, especially during the onset of remodelling where it is believed they may be responsible for osteoclast recruitment and/or differentiation. The aim of this study was to look for further evidence of mast cell (MC) involvement in pathological bone resorption. MC activity was assessed in tissue sections of osteolytic conditions including Paget’s disease of bone, rheumatoid arthritis and fibrous dysplasia together with several benign and malignant bone tumours. MCs were identified by toluidine blue staining and by immunostaining with a commercial antibody against MC tryptase.

Extensive infiltration of mast cells was observed in fibrous dysplasia, rheumatoid arthritis and Paget’s disease of bone and mast cell accumulation was seen at the bone resorbing margin of a number of enlarging bone tumours including osteosarcoma, giant cell tumour of bone, osteoma and osteoid osteoma.

MCs, along with other inflammatory cells, are known to accumulate at the margins of soft tissue tumours where they are thought to promote tumour growth. The current findings are consistent with a similar role for mast cells in the primary bone tumours examined. In each of the conditions studied, an additional role for MC may be that of promoting bone lysis. MC are known to contain numerous factors including TNF-alpha and IL-1, which are potent stimulators of osteoclast formation and activity.

It is concluded that MCs may contribute to the fibrosis, angiogenesis and increased bone resorption seen in certain metabolic bone diseases. MC activity may also be an important factor contributing to the lysis that occurs in numerous other pathological situations including at the margins of aggressive primary bone tumours and skeletal metastases, leading to the expansion of these lesions.

1. The use of the Metals Research Macrotome for cutting 100 μ thick sections of fresh, unfixed specimens of arthritic human femoral heads and normal goat condyles is described.

2. A technique for isolating living cells from these slices by decalcification followed by enzymic digestion is reported.

3. The microscopic appearances of the fresh slices, the decalcified slices and the isolated cells as seen by incident or transmitted fluorescent lighting, by phase-contrast microscopy, by scanning electron microscopy and by histological and cytological techniques are illustrated.

4. These techniques might be applicable to the examination of biopsy specimens of pathological bone or to basic research on bone cells.

1. Methods for culturing cells isolated from slices of arthritic human or normal mammalian cancellous bone are described.

2. The capacity of the cultured cells to take up and hydroxylate labelled proline has been investigated.

3. Sections of the partially decalcified bone and of the isolated cells have been examined by transmission electron microscopy.

4. The possible significance of the results and observations are discussed. We are deeply grateful to Dame Janet Vaughan, who very kindly read this manuscript and made several valuable suggestions and criticisms. We are much obliged to Dr Sylvia Fitton-Jackson for her advice on the techniques of tissue culture and for giving us the composition of her chemically defined medium. Dr Palfrey kindly allowed one of us, M. J. Dickens, to learn transmission electron microscopy in his department at St Thomas's Hospital Medical School under the expert tuition of Mr G. Maxwell. Mr R. Hockhan and Mr M. Hepburn of the University of Surrey Structural Studies Unit helpfully instructed in the operation of the transmission electron microscope. Our special thanks are due to Mr E. P. Morris for his competent and enthusiastic technical assistance.