Controversies in orthopaedic oncology.

Chondrosarcoma is the second most common surgically treated primary bone sarcoma. Despite a large number of scientific papers in the literature, there is still significant controversy about diagnostics, treatment of the primary tumour, subtypes, and complications. Therefore, consensus on its day-to-day treatment decisions is needed. In January 2024, the Birmingham Orthopaedic Oncology Meeting (BOOM) attempted to gain global consensus from 300 delegates from over 50 countries. The meeting focused on these critical areas and aimed to generate consensus statements based on evidence amalgamation and expert opinion from diverse geographical regions. In parallel, periprosthetic joint infection (PJI) in oncological reconstructions poses unique challenges due to factors such as adjuvant treatments, large exposures, and the complexity of surgery. The meeting debated two-stage revisions, antibiotic prophylaxis, managing acute PJI in patients undergoing chemotherapy, and defining the best strategies for wound management and allograft reconstruction. The objectives of the meeting extended beyond resolving immediate controversies. It sought to foster global collaboration among specialists attending the meeting, and to encourage future research projects to address unsolved dilemmas. By highlighting areas of disagreement and promoting collaborative research endeavours, this initiative aims to enhance treatment standards and potentially improve outcomes for patients globally. This paper sets out some of the controversies and questions that were debated in the meeting.

Collared Cementless Femoral Components Reduce the Revision Rates in Primary Total Hip Arthroplasty Using the Direct Anterior Approach: An Australian Orthopaedic Association National Joint Replacement Registry Study.

BACKGROUND: An increased risk of periprosthetic fracture and aseptic loosening is reported when the direct anterior approach (DAA) is used for total hip arthroplasty (THA), especially with cementless implants. We assessed the rate of revision comparing collared and collarless femoral stems when using the DAA for THA. METHODS: We used data from the Australian Orthopaedic Association National Joint Replacement Registry for primary THA for osteoarthritis inserted with the DAA between January 2015 and December 2022. There were 48,567 THAs that used the DAA (26,690 collarless cementless, 10,161 collared cementless, and 11,716 cemented). Cumulative percent revision was calculated for all-cause revision, revision for periprosthetic femoral fractures, and aseptic femoral stem loosening. Cox proportional hazard ratios [HRs] were used to compare the revision of collared and collarless cementless stems. We also compared collared cementless stems and cemented stems. RESULTS: A higher rate of all-cause revision within 3 months of surgery was observed with collarless compared to collared cementless implants (HR: 1.99 [95% confidence interval (CI), 1.56 to 2.54]; P < .001). Similarly, collarless cementless implants were associated with a greater rate of revision for fracture in the first 6 months (HR: 2.90 [95% CI, 1.89 to 4.45]; P < .001) and after 6 months (HR 10.04 [95% CI 1.38 to 73.21]; P = .02), as well as an increased rate of revision for aseptic loosening after 2 years (HR: 5.76 [95% CI, 1.81 to 18.28], P = .003). Collared cementless and cemented stems performed similarly. CONCLUSION: Collared stems were associated with a reduced rate of all-cause revision for cementless THA performed via the DAA. The reduction in risk may be due to protection from periprosthetic femoral fracture and aseptic loosening.

NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential.

Tissue-engineered implants for bone regeneration require consideration regarding their mineralization and vascularization capacity. Different geometries, such as biomimetic designs and lattices, can influence the mechanical properties and the vascularization capacity of bone-mimicking implants. Negative Embodied Sacrificial Template 3D (NEST3D) printing is a versatile technique across a wide range of materials that enables the production of bone-mimicking scaffolds. In this study, different scaffold motifs (logpile, Voronoi, and trabecular bone) were fabricated via NEST3D printing in polycaprolactone to determine the effect of geometrical design on stiffness (10.44 ± 6.71, 12.61 ± 5.71, and 25.93 ± 4.16 MPa, respectively) and vascularization. The same designs, in a polycaprolactone scaffold only, or when combined with gelatin methacryloyl, were then assessed for their ability to allow the infiltration of blood vessels in a chick chorioallantoic membrane (CAM) assay, a cost-effective and time-efficient in ovo assay to assess vascularization. Our findings showed that gelatin methacrylolyl alone did not allow new chorioallantoic membrane tissue or blood vessels to infiltrate within its structure. However, polycaprolactone on its own or when combined with gelatin methacrylolyl allowed tissue and vessel infiltration in all scaffold designs. The trabecular bone design showed the greatest mineralized matrix production over the three designs tested. This reinforces our hypothesis that both biomaterial choice and scaffold motifs are crucial components for a bone-mimicking scaffold.

Negative Printing for the Reinforcement of In Situ Tissue-Engineered Cartilage.

In the realm of in situ cartilage engineering, the targeted delivery of both cells and hydrogel materials to the site of a defect serves to directly stimulate chondral repair. Although the in situ application of stem cell-laden soft hydrogels to tissue defects holds great promise for cartilage regeneration, a significant challenge lies in overcoming the inherent limitation of these soft hydrogels, which must attain mechanical properties akin to the native tissue to withstand physiological loading. We therefore developed a system where a gelatin methacryloyl hydrogel laden with human adipose-derived mesenchymal stem cells is combined with a secondary structure to provide bulk mechanical reinforcement. In this study, we used the negative embodied sacrificial template 3D printing technique to generate eight different lattice-based reinforcement structures made of polycaprolactone, which ranged in porosity from 80% to 90% with stiffnesses from 28 ± 5 kPa to 2853 ± 236 kPa. The most promising of these designs, the hex prism edge, was combined with the cellular hydrogel and retained a stable stiffness over 41 days of chondrogenic differentiation. There was no significant difference between the hydrogel-only and hydrogel scaffold group in the sulfated glycosaminoglycan production (340.46 ± 13.32 µg and 338.92 ± 47.33 µg, respectively) or Type II Collagen gene expression. As such, the use of negative printing represents a promising solution for the integration of bulk reinforcement without losing the ability to produce new chondrogenic matrix.

Clinical activity in general practice prior to sarcoma diagnosis: Australian cohort study.

Background Increased time-to-diagnosis in sarcoma is associated with poor prognosis and patient outcomes. Research is needed to identify if opportunities to expedite the diagnosis of sarcoma in general practice (GP) exist. Aim To examine pre-diagnostic GP clinical activity prior to sarcoma diagnosis. Design and Setting An Australian retrospective cohort study using hospital registry data (Australian Comprehensive Cancer Outcomes and Research Database) linked to two primary care datasets (Patron and MedicineInsight). Method The frequency of GP healthcare utilisation events (GP attendances, prescriptions, blood test and imaging requests) were compared in 377 soft tissue sarcoma (STS) and 64 bone sarcoma (BS) patients in the year pre-diagnosis. Poisson regression models were used to calculate monthly incidence rates and rate ratios (IRR) for the 24 months pre-diagnosis and estimate inflection points for when healthcare use starts to increase from baseline. Results In the six months pre-diagnosis sarcoma patients had a median of 3-4 GP attendances, a third had a GP imaging request (33% BS and 36% STS), and one in five had multiple imaging requests (19% BS and 21% STS). GP imaging requests progressively increased up to 8-fold from 6 months prior to sarcoma diagnosis (IRR 8.43 95%CI 3.92-18.15, p<0.001) and GP attendances increased from 3 months pre-diagnosis. Conclusion Sarcoma patients have increased GP clinical activity from 6 months pre-diagnosis, indicating a diagnostic window where potential opportunities exist for earlier diagnosis. Interventions to help identify patients and promote appropriate use of imaging and direct specialist centre referrals could improve earlier diagnosis and patient outcomes.

Bridging bench to body: ex vivo models to understand articular cartilage repair.

With little to no ability to self-regenerate, human cartilage defects of the knee remain a major clinical challenge. Tissue engineering strategies include delivering specific types of cells and biomaterials to the injured cartilage for restoration of architecture and function. Pre-clinical models to test the efficacy of the therapies come with high costs and ethical issues, and imperfect prediction of performance in humans. Ex vivo models represent an alternative avenue to trial cartilage tissue engineering. Defined as viable explanted cartilage samples, ex vivo models can be cultured with a cell-laden biomaterial or tissue-engineered construct to evaluate cartilage repair. Though human and animal ex vivo models are currently used in the field, there is a need for alternative methods to assess the strength of integration, to increase throughput and manage variability and to optimise and standardise culture conditions, enhancing the utility of these models overall.

What to choose in bone tumour resections? Patient specific instrumentation versus surgical navigation: a systematic review.

Patient specific instrumentation (PSI) and intraoperative surgical navigation (SN) can significantly help in achieving wide oncological margins while sparing bone stock in bone tumour resections. This is a systematic review aimed to compare the two techniques on oncological and functional results, preoperative time for surgical planning, surgical intraoperative time, intraoperative technical complications and learning curve. The protocol was registered in PROSPERO database (CRD42023422065). 1613 papers were identified and 81 matched criteria for PRISMA inclusion and eligibility. PSI and SN showed similar results in margins (0-19% positive margins rate), bone cut accuracy (0.3-4 mm of error from the planned), local recurrence and functional reconstruction scores (MSTS 81-97%) for both long bones and pelvis, achieving better results compared to free hand resections. A planned bone margin from tumour of at least 5 mm was safe for bone resections, but soft tissue margin couldn't be planned when the tumour invaded soft tissues. Moreover, long osteotomies, homogenous bone topology and restricted working spaces reduced accuracy of both techniques, but SN can provide a second check. In urgent cases, SN is more indicated to avoid PSI planning and production time (2-4 weeks), while PSI has the advantage of less intraoperative using time (1-5 min vs 15-65 min). Finally, they deemed similar technical intraoperative complications rate and demanding learning curve. Overall, both techniques present advantages and drawbacks. They must be considered for the optimal choice based on the specific case. In the future, robotic-assisted resections and augmented reality might solve the downsides of PSI and SN becoming the main actors of bone tumour surgery.

Arithmetic hip knee angle measurement on long leg radiograph versus computed tomography-inter-observer and intra-observer reliability.

BACKGROUND: Pre-operative alignment is important for knee procedures including total knee arthroplasty (TKA), especially when considering alternative alignments. The arithmetic Hip Knee Angle (aHKA) is a measure of coronal alignment calculated using the medial proximal tibial (MPTA) and lateral distal femoral angles (LDFA). Traditionally, aHKA is measured on long leg radiographs (LLR). This study assesses the reproducibility of aHKA measurement on LLR and robotic-assisted TKA planning CT. METHODS: Sixty-eight TKA patients with pre-operative LLR and planning CTs were included. Three observers measured the LDFA, MPTA and aHKA three times on each modality and intra-observer and inter-observer reliability was calculated. Statistical analysis was undertaken with Pearson's r and the Bland-Altman test. RESULTS: Mean intra-observer coefficient of repeatability (COR) for LLR vs. CT: MPTA 3.50° vs. 1.73°, LDFA 2.93° vs. 2.00° and aHKA 2.88° vs. 2.57° for CT. Inter-observer COR for LLR vs. CT: MPTA 2.74° vs. 1.28°, LDFA 2.31° vs. 1.92°, aHKA 3.56° vs. 2.00°. Mean intra-observer Pearson's r for MPTA was 0.93 for LLR and 0.94 for CT, LDFA 0.90 for LLR and 0.91 for CT and aHKA 0.92 for LLR and 0.94 for CT. Inter-observer Pearson's r for LLR compared to CT: MPTA 0.93 vs. 0.97, LDFA 0.91 vs. 0.90, aHKA 0.91 and 0.95. CONCLUSION: When compared to LLR, CT measurements of MPTA, LDFA and aHKA are more reproducible and have a good correlation with LLR measurement. CT overcomes difficulties with positioning, rotation, habitus and contractures when assessing coronal plane alignment and may obviate the need for LLRs.

Oncologic outcomes in myxofibrosarcomas: the role of a multidisciplinary approach and surgical resection margins.

BACKGROUNDS: Myxofibrosarcomas (MFS) are malignant soft tissue sarcomas with an infiltrative growth pattern and propensity for local recurrence(LR).We aimed to assess our management of MFS and make recommendations about the role of a multidisciplinary team approach and margin widths. METHODS: Fifty-seven patients were identified with MFS treated at a single sarcoma centre between 1998 and 2020. Patients were stratified based on whether they presented for a planned resection (59.6%) or after an unplanned resection (40.4%) performed at a non-specialized facility. All patients underwent radiotherapy before definitive surgery. RESULTS: 73.7% underwent a combined onco-plastic approach. The 5 year LRFS rate was 78.2% (84.4%, planned, versus 70.1%, unplanned, P = 0.194) and found comparable oncological outcomes between the planned and unplanned groups for the 5 year metastasis free survival (74.5% versus 86.1%, P = 0.257), disease free survival (70.1% versus 72.4%, P = 0.677), and Overall Survival (64.5% versus 75.9%, P = 0.950). Margin width ≥ 2 cm was obtained in 84.2% of cases and improved local control (HR = 0.22; 95% CI 0.06-0.81; P = 0.023), metastasis (HR = 0.24; 95% CI 0.07-0.80; P = 0.019) and mortality rates (HR = 0.23; 95% CI 0.09, 0.61; P = 0.003) compared to <2 cm. Margin width > 3 cm did not further affect oncological outcomes. CONCLUSION: Our study shows that a multidisciplinary team approach allows the achievement of low local recurrence rate and good oncological outcomes of myxofibrosarcomas, regardless of presentation status. We recommend a minimum of 2 cm margin width.

Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy.

BACKGROUND: Articular cartilage repair using implantable photocrosslinkable hydrogels laden with chondrogenic cells, represents a promising in situ cartilage engineering approach for surgical treatment. The development of a surgical procedure requires a minimal viable product optimized for the clinical scenario. In our previous work we demonstrated how gelatin based photocrosslinkable hydrogels in combination with infrapatellar derived stem cells allow the production of neocartilage in vitro. In this study, we aim to optimize the critical facets of the in situ cartilage engineering therapy: the cell source, the cell isolation methodology, the cell expansion protocol, the cell number, and the delivery approach. METHODS: We evaluated the impact of the critical facets of the cell-laden hydrogel therapy in vitro to define an optimized protocol that was then used in a rabbit model of cartilage repair. We performed cells counting and immunophenotype analyses, chondrogenic potential evaluation via immunostaining and gene expression, extrusion test analysis of the photocrosslinkable hydrogel, and clinical assessment of cartilage repair using macroscopic and microscopic scores. RESULTS: We identified the adipose derived stem cells as the most chondrogenic cells source within the knee joint. We then devised a minimally manipulated stem cell isolation procedure that allows a chondrogenic population to be obtained in only 85 minutes. We found that cell expansion prior to chondrogenesis can be reduced to 5 days after the isolation procedure. We characterized that at least 5 million of cells/ml is needed in the photocrosslinkable hydrogel to successfully trigger the production of neocartilage. The maximum repairable defect was calculated based on the correlation between the number of cells retrievable with the rapid isolation followed by 5-day non-passaged expansion phase, and the minimum chondrogenic concentration in photocrosslinkable hydrogel. We next optimized the delivery parameters of the cell-laden hydrogel therapy. Finally, using the optimized procedure for in situ tissue engineering, we scored superior cartilage repair when compared to the gold standard microfracture approach. CONCLUSION: This study demonstrates the possibility to repair a critical size articular cartilage defect by means of a surgical streamlined procedure with optimized conditions.

Within or Without You? A Perspective Comparing In Situ and Ex Situ Tissue Engineering Strategies for Articular Cartilage Repair.

Human articular cartilage has a poor ability to self-repair, meaning small injuries often lead to osteoarthritis, a painful and debilitating condition which is a major contributor to the global burden of disease. Existing clinical strategies generally do not regenerate hyaline type cartilage, motivating research toward tissue engineering solutions. Prospective cartilage tissue engineering therapies can be placed into two broad categories: i) Ex situ strategies, where cartilage tissue constructs are engineered in the lab prior to implantation and ii) in situ strategies, where cells and/or a bioscaffold are delivered to the defect site to stimulate chondral repair directly. While commonalities exist between these two approaches, the core point of distinction-whether chondrogenesis primarily occurs "within" or "without" (outside) the body-can dictate many aspects of the treatment. This difference influences decisions around cell selection, the biomaterials formulation and the surgical implantation procedure, the processes of tissue integration and maturation, as well as, the prospects for regulatory clearance and clinical translation. Here, ex situ and in situ cartilage engineering strategies are compared: Highlighting their respective challenges, opportunities, and prospects on their translational pathways toward long term human cartilage repair.

Standardised quantitative ultrasound imaging approach for the contact-less three-dimensional analysis of neocartilage formation in hydrogel-based bioscaffolds.

In this work we present a standardised quantitative ultrasound imaging (SQUI) approach for the non-destructive three-dimensional imaging and quantification of cartilage formation in hydrogel based bioscaffolds. The standardised concept involves the processing of ultrasound backscatter data with respect to an acellular phantom in combination with the compensation of sound speed mismatch diffraction effects between the bioscaffold and the phantom. As a proof-of-concept, the SQUI approach was tested on a variety of bioscaffolds with varying degree of neocartilage formation. These were composed of Gelatine Methacryloyl (GelMA) hydrogels laden with human adipose-derived stem cells (hADSCs). These were cultured under chondrogenic stimulation following a previously established protocol, where the degree of the neocartilage formation was modulated using different GelMA network densities (6, 8, 10 % w/v) and culture time (0, 14, 28 days). Using the SQUI approach we were able to detect marked acoustic and morphological changes occurring in the bioscaffolds a result of their different chondrogenic outcome. We defined an acoustic neocartilage indicator, the sonomarker, for the selective imaging and quantification of neocartilage formation. The sonomarker, of backscatter intensity logIBC -2.4, was found to correlate with data obtained via standard destructive bioassays. The ultrasonic evaluation of human specimens confirmed the sonomarker as a relevant intensity, although it was found to shift to higher intensity values in proportion to the cartilage condition as inferred from sound speed measurements. This study demonstrates the potential of the SQUI approach for the realization of non-destructive analysis of cartilage regeneration over-time. STATEMENT OF SIGNIFICANCE: As tissue engineering strategies for neocartilage regeneration evolve towards clinical implementation, alternative characterisation approaches that allow the non-destructive monitoring of extracellular matrix formation in implantable hydrogel based bioscaffolds are needed. In this work we present an innovative standardized quantitative ultrasound imaging (SQUI) approach that allows the non-destructive, volumetric, and quantitative evaluation of neocartilage formation in hydrogel based bioscaffolds. The standardised concept aims to provide a robust approach that accounts for the dynamic changes occurring during the conversion from a cellular bioscaffold towards the formation of a neocartilage construct. We believe that the SQUI approach will be of great benefit for the evaluation of constructs developing neocartilage, not only for in-vitro applications but also potentially applicable to in-vivo applications.

Molecular Pathogenesis of Sporadic Desmoid Tumours and Its Implications for Novel Therapies: A Systematised Narrative Review.

Sporadic desmoid-type fibromatosis is a rare, fibroblastic soft-tissue neoplasm with local aggressiveness but no metastatic potential. Aberrant Wnt/ß-catenin signalling has been extensively linked to desmoid pathogenesis, although little is known about other molecular drivers and no established treatment approach exists. We aimed to summarise the current literature regarding the molecular pathogenesis of sporadic desmoid-type fibromatosis and to discuss the effects of both current and emerging novel therapies targeting these mechanisms. A literature search was conducted of MEDLINE® ALL and EMBASE databases for published studies (2000-August 2021) using keywords related to 'fibromatosis aggressive', 'immunohistochemistry', 'polymerase chain reaction' and 'mutation'. Articles were included if they examined the role of proteins in sporadic or extra-abdominal human desmoid-type fibromatosis pathogenesis. Searching identified 1684 articles. Following duplicate removal and eligibility screening, 36 were identified. After a full-text screen, 22 were included in the final review. At least 47% of desmoid-type fibromatosis cases displayed aberrant ß-catenin immunoreactivity amongst ten studies. Cyclin D1 overexpression occurred in at least 40% of cases across five studies. Six studies reported oestrogen receptor-ß expression with a range of 7.4-90%. Three studies implicated matrix metalloproteinases, with one study demonstrating vascular endothelial growth factor overexpression. One study explored the positive relationship between cyclooxygenase-2 and platelet-derived growth factor receptor-ß. Aberrant Wnt/ß-catenin signalling is a well-established pathogenic driver that may be targeted via downstream modulation. Growth factor signalling is best appreciated through the clinical trial effects of multi-targeted tyrosine kinase inhibitors, whilst oestrogen receptor expression data may only offer a superficial insight into oestrogen signalling. Finally, the tumour microenvironment presents multiple potential novel therapeutic targets.

Sporadic desmoid tumours are rare soft-tissue neoplasms that arise from connective tissues in the chest wall, head, neck and limbs. Whilst lacking metastatic potential, uncertainty surrounding their locally aggressive growth and unpredictable recurrence complicates treatment approaches. At the molecular level, alterations in the Wnt/ß-catenin signalling pathway, a fundamental coordinator of cell growth and development, have been strongly linked to desmoid tumour development. Beyond this, however, little is known about other molecular drivers. In the case of progressive or life-threatening disease, complex treatment decisions are made regarding the use of surgery, radiotherapy or systemic treatment modalities. Of the targeted systemic therapies, a lack of comparative clinical studies further complicates medical treatment decision making as no definitive treatment approach exists. Therefore, this review aimed to summarise the literature regarding the molecular drivers of desmoid tumour pathogenesis and to discuss the current and emerging novel therapies targeting such mechanisms. Utilising findings from human desmoid tissue samples, we present the rationale for targeting downstream mediators of the central Wnt/ß-catenin pathway and outline potential treatment targets in the tumour microenvironment. We also highlight the knowledge gained from clinical drug trials targeting desmoid growth factor signalling and present the potentially superficial insight provided by oestrogen receptor expression profiles on the role of oestrogen signalling in desmoid pathogenesis. In doing so, this work may assist in the eventual development of an evidence-based treatment approach for sporadic desmoid tumours.

The Challenge of Cartilage Integration: Understanding a Major Barrier to Chondral Repair.

Articular cartilage defects caused by injury frequently lead to osteoarthritis, a painful and costly disease. Despite widely used surgical methods to treat articular cartilage defects and a plethora of research into regenerative strategies as treatments, long-term clinical outcomes are not satisfactory. Failure to integrate repair tissue with native cartilage is a recurring issue in surgical and tissue-engineered strategies, seeing eventual degradation of the regenerated or surrounding tissue. This review delves into the current understanding of why continuous and robust integration with native cartilage is so difficult to achieve. Both the intrinsic limitations of chondrocytes to remodel injured cartilage, and the significant challenges posed by a compromised biomechanical environment are described. Recent scaffold and cell-based techniques to repair cartilage are also discussed, and limitations of existing methods to evaluate integrative repair. In particular, the importance of evaluating the mechanical integrity of the interface between native and repair tissue is highlighted as a meaningful assessment of any strategy to repair this load-bearing tissue. Impact statement The failure to integrate grafts or biomaterials with native cartilage is a major barrier to cartilage repair. An in-depth understanding of the reasons cartilage integration remains a challenge is required to inform cartilage repair strategies. In particular, this review highlights that integration of cartilage repair strategies is frequently assessed in terms of the continuity of tissue, but not the mechanical integrity. Given the load-bearing nature of cartilage, evaluating integration in terms of interfacial strength is essential to assessing the potential success of cartilage repair methods.

3D Printed Multiphasic Scaffolds for Osteochondral Repair: Challenges and Opportunities.

Osteochondral (OC) defects are debilitating joint injuries characterized by the loss of full thickness articular cartilage along with the underlying calcified cartilage through to the subchondral bone. While current surgical treatments can provide some relief from pain, none can fully repair all the components of the OC unit and restore its native function. Engineering OC tissue is challenging due to the presence of the three distinct tissue regions. Recent advances in additive manufacturing provide unprecedented control over the internal microstructure of bioscaffolds, the patterning of growth factors and the encapsulation of potentially regenerative cells. These developments are ushering in a new paradigm of 'multiphasic' scaffold designs in which the optimal micro-environment for each tissue region is individually crafted. Although the adoption of these techniques provides new opportunities in OC research, it also introduces challenges, such as creating tissue interfaces, integrating multiple fabrication techniques and co-culturing different cells within the same construct. This review captures the considerations and capabilities in developing 3D printed OC scaffolds, including materials, fabrication techniques, mechanical function, biological components and design.

Microencapsulation of growth factors by microfluidic system.

The encapsulation of growth factors is an important component of tissue engineer- ing. Using microspheres is a convenient approach in which the dose of factors can be regulated by increasing or decreasing the number of encapsulated microspheres. Moreover, microspheres offer the possibility of delivering the growth factors directly to the target site. However, the fabrication of microspheres by traditional emulsion methods is largely variable due to the experimental procedure. We have developed a protocol using a commercially available microfluidic system that allows formation of tunable particle-size droplets loaded with growth factors. The methodology includes a guide for preparing an alginate-growth factors solution followed by the specific set-up needed for using the microfluidic system to form the microspheres. The pro- cedure also includes a unique post-crosslinking process without pH modification. These methods allow the preservation of integrity and bioactivity of the growth factors tested (BMP-6 and TGFß -3) and their subsequent sustained delivery.•The protocol can be tuned to form particles of various sizes.•The gentle post-crosslinking process allows conformational integrity of various bioactive molecules.

Microbial Transglutaminase Improves ex vivo Adhesion of Gelatin Methacryloyl Hydrogels to Human Cartilage.

Current surgical techniques to treat articular cartilage defects fail to produce a satisfactory long-term repair of the tissue. Regenerative approaches show promise in their ability to generate hyaline cartilage using biomaterials in combination with stem cells. However, the difficulty of seamlessly integrating the newly generated cartilage with the surrounding tissue remains a likely cause of long-term failure. To begin to address this integration issue, our strategy exploits a biological enzyme (microbial transglutaminase) to effect bioadhesion of a gelatin methacryloyl implant to host tissue. Mechanical characterization of the bioadhesive material shows that enzymatic crosslinking is compatible with photocrosslinking, allowing for a dual-crosslinked system with improved mechanical properties, and a slower degradation rate. Biocompatibility is illustrated with a 3D study of the metabolic activity of encapsulated human adipose derived stem cells. Furthermore, enzymatic crosslinking induced by transglutaminase is not prevented by the presence of cells, as measured by the bulk modulus of the material. Adhesion to human cartilage is demonstrated ex vivo with a significant increase in adhesive strength (5.82 ± 1.4 kPa as compared to 2.87 ± 0.9 kPa, p < 0.01) due to the addition of transglutaminase. For the first time, we have characterized a bioadhesive material composed of microbial transglutaminase and GelMA that can encapsulate cells, be photo crosslinked, and bond to host cartilage, taking a step toward the integration of regenerative implants.

FLASH: Fluorescently LAbelled Sensitive Hydrogel to monitor bioscaffolds degradation during neocartilage generation.

Regenerative therapies based on photocrosslinkable hydrogels and stem cells are of growing interest in the field of cartilage repair. Cell-mediated degradation is critical for the successful clinical translation of implanted hydrogels. However, characterising cell-mediated degradation, while simultaneously monitoring the deposition of a distinct new matrix, remains a major challenge. In this study we generated a Fluorescently LAbelled Sensitive Hydrogel (FLASH) to correlate the degradation of a hydrogel bioscaffold with neocartilage formation. Gelatine Methacryloyl (GelMA) was covalently bound to the FITC fluorophore to generate FLASH and bioscaffolds were produced by casting different concentrations of FLASH GelMA, with and without human adipose-derived stem cells (hADSCs) undergoing chondrogenesis. The loss of fluorescence from FLASH bioscaffolds was correlated with changes in mechanical properties, expression of chondrogenic markers and accumulation of a cartilaginous extracellular matrix. The ability of the system to be used as a sensor to monitor bioscaffold degradability during chondrogenesis was evaluated in vitro, in a human ex vivo model of cartilage repair and in a full chondral defect in vivo rabbit model. This study represents a step towards the generation of a high throughput monitoring system to evaluate de novo cartilage formation in tissue engineering therapies.

Human Stem Cell Based Tissue Engineering for In Vivo Cartilage Repair: A Systematic Review.

Pure chondral defects represent the most clinically significant articular cartilage injuries. To inform the development of clinically suitable tissue-engineering strategies for chondral repair using cells from a human patient, the combination of human stem cells (HSCs), biomaterial scaffolds, and growth factors has been widely harnessed in preclinical animal models. Due to the large heterogeneity in study designs and outcome reporting in such studies, we aimed to systematically review literature pertaining to HSC based tissue engineering strategies in animal models of chondral repair such that trends may be identified and the utility of HSCs in chondral repair can be elucidated. An extensive search strategy was carried out through PubMed, MEDLINE, and EMBASE databases to identify relevant studies. Initially the title and abstract of 787 studies were screened after which inclusion and exclusion criteria sorted 56 studies for full-text evaluation. Following full text review, a final number of 22 articles were included. Out of 22 included studies, 16 used scaffold implantation, 2 used cell pellet implantation, and 4 used intra-articular injection to administer HSCs to the region of chondral defects. HSC-containing implants outperformed scaffold-only or untreated control groups in both large and small animals for chondral regeneration. Umbilical cord mesenchymal stem cells and hyaluronic acid-containing scaffolds emerged as popular stem cell and scaffold choices, respectively. However, the short analysis timepoints post cell implantation was a key limitation in many studies. This review highlights the versatility of HSCs in achieving chondral regeneration in vivo and the enhancement of chondral repair through the selection of appropriate three-dimensional scaffolds and growth factors which are essential to support cell growth, attachment, migration, and extracellular matrix synthesis. Considerable heterogeneity exists in outcome reporting, and only one article reported biomechanical evaluation of neocartilage. Standardized outcome reporting systems that include comprehensive biomechanical testing protocols should be utilized in future in vivo studies of cartilage tissue engineering as the biomechanical quality of neocartilage is of great functional significance.