There is currently no cure for osteoarthritis (OA), although there are ways to manage it, but most require quite invasive surgeries. There is a resident mesenchymal progenitor cell (MPC) population within the synovial membrane of the joint that have the ability to differentiate into bone, fat, and cartilage. We hypothesise that in vivo and in vitro cell surface marker expression comparisons of the MPCs can determine which population has the highest chondrogenic capacity and is best suited for future clinical trials. Method optimisation protocol: Synovial biopsies (2 or 5mm) were obtained from patients undergoing surgery. The biopsies were digested in either collagenase type I, IA, IV or II at a concentration of 0.5 or 1.0 mg/mL. Digestion was conducted at 37°C for 30, 60, 90 or 120min. To assay for the number of MPCs obtained, the cell suspension was stained with CD90 (a synovial MPC marker) and magnetically purified. The purified cells were then assayed by flow cytometry (Co-stained with a live/dead cell marker, BV510) or bright-field microscopy. Study protocol: Synovial tissues were digested in type IV collagenase for two hours to obtain a single cell suspension. The cells were subsequently stained with mesenchymal stem cell markers, including CD 90, CD 271, CD 44, CD73, and CD105, a macrophage marker, CD68. The macrophages were excluded and the remaining cells were index sorted into 96-well plates. The cells were expanded, and underwent 21-day chondrogenic, adipogenic, and osteogenic differentiation. Differentiation was assayed using RT-qPCR and histological methods. Additionally, the cells were re-analysed for marker expression after culturing. Optimisation: Synovial biopsies of 5mm produced a greater number of live CD90+ cells than 2mm biopsies. It was observed that type IV collagenase at 1mg/ML treatment for 120 min (hip) and 90 min (knee) obtained the greatest number of CD90+ MPCs from the synovium. Results: A single cell was isolated from an OA hip biopsy and was positive for the markers CD90, CD44, CD73, and negative for the markers CD68, CD271, CD105. Following differentiation, PCR analysis suggested that the cell line was able to differentiate into chondrocytes and adipocytes, but not osteoblasts. Histology data agreed with the PCR data with the adipocytes and chondrocytes having positive staining, whereas the osteoblasts were negative. FACS analysis following proliferation showed that the expression in vivo versus in vitro was the same except CD105 that became positive after proliferation in vitro. MPCs express cell surface markers that provide information as to populations have the best cartilage regeneration abilities. By determining the properties of the MPCs in OA hips that allow for better chondrogenic differentiation abilities in vitro, selecting the optimal cells for regenerating cartilage can be done more efficiently for novel cell therapies for OA

Numerous investigators have described osteogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications, such as operative treatment of non-unions and regeneration of critical bone defects. Therefore, the primary goal of this research endeavor is to achieve osteogenic differentiation of ADSC. Previous work has already demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for healing critical bone defects. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our stem cell population in order to direct their differentiation into the osteoblastic lineage.

We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized as a reporter gene to screen and enrich the ADSC population for only those stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20 days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin. Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCR for alkaline phosphatase gene expression as a biomarker for the osteoblastic lineage.

Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous osteoblasts in vitro for future implantation at the site of critical bone defects. This method of attaining osseous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance of signaling mediated by the BMP receptor 1A during osteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenic differentiation potential of these stem cells under hydrostatic and fluid flow shearing mechanical loads.

Orthopaedic surgery is a practical surgical specialization field, the exit exam for registrars remains written and oral. Despite logbook evaluation and surgical work-based assessments, the question remains: can registrars perform elective surgery upon qualification? In South Africa, obstacles to elective surgical training include the trauma workload, financial constraints, fellowships and the Covid pandemic. In hip and knee arthroplasty, new approaches like the direct anterior approach (DAA) and robotic-assisted knee surgery also contributed to the dilution of cases available for registrar training. There are concerns that orthopaedic registrars do not perform enough cases to achieve surgical proficiency. Review of the last 4 years of registrar logbooks in hip and knee arthroplasty surgery performed in a single tertiary academic hospital in South Africa. We included all primary total hip replacements (THR), total knee replacements (TKR) and hemiarthroplasties (HA) done for neck of femur (NOF) fractures between 1 April 2019 and 30 March 2023. Differentiation between registrar assisting, registrar performing with consultant supervision and registrar performing independent surgery was done. 990 hip arthroplasties (472 Primary THR, 216 NOF THR, 302 NOF HA) and 316 Primary TKR were performed during the study period. In primary elective THR the posterior approach was dominant and used in 76% of cases. In NOF THA the DAA was dominant used in 98% of cases. Primary TKR robotic-assisted technologies was used in 27% (n=94) cases. Registrars as the primary surgeon were the highest in NOF THA at 70% of cases and the lowest performing TKR at 25%. During 3-month rotations, an average registrar performed 12 (2 TKR and 10 THR) and assisted in 35 (10 TKR and 25 THR) cases. Despite the large number of arthroplasties operations being performed over the last 4-year period, the surgical cases done by registrars are below, the proposed minimal cases to provide surgical proficiency during their training period

Aim. Treatment algorithms for fracture-related nonunion depend on the presence or absence of bacterial infection. However, the manifestation of septic nonunion varies. Low-grade infections, unlike manifest infections, lack clinical signs of infection and present similarly to aseptic nonunion. The clinical importance of low-grade infection in nonunion is not entirely clear. Therefore, the aim of this study was to evaluate the clinical relevance of low-grade infection in the development and management of femoral or tibial nonunion. Method. A prospective, multicenter clinical study enrolled patients with nonunion and regular healed fractures. Preoperatively, complete blood count without differential, C-reactive protein (CRP), and procalcitonin were obtained, clinical signs of infection were recorded, and a suspected septic or aseptic diagnosis was made based on history and clinical examination. During surgical nonunion revision or routine implant removal, tissue samples were collected for microbiology and histopathology, and osteosynthesis material for sonication. Nonunion patients were followed for 12 months. Definitive diagnosis of “septic” or “aseptic” nonunion was made according to diagnostic criteria for fracture-related infection, considering the results of any further revision surgery during follow-up. Results. 34 patients with regular healed fractures were included. 62 nonunion patients were diagnosed as aseptic, 22 with manifest, and 23 with low-grade infection. The positive predictive value was 88% and the negative predictive value 72% for the suspected diagnosis. The nonunion groups had significantly higher CRP levels than the regular healer group. Differentiation between septic and aseptic nonunion based on blood values was not possible. Low-grade infection demonstrated less frequently histopathologic signs of infection than manifest infection (22% vs. 50%, p=0.048), with 15% of regular healers having histopathologic signs of infection. Cutibacterium acnes was less present in manifest compared to low-grade infection (p=0.042). Healing rates for septic nonunion involving C. acnes were significantly lower for manifest infection (20%) than for low-grade infection (100%, p=0.002). Patients with low-grade infection were treated with systemic antibiotics less frequently than patients with manifest infection (p=0.026), with no significant difference in healing rate (83% vs. 64%), which was slightly lower for low-grade infection than for aseptic nonunion (90%). Conclusions. Low-grade infections play a significant role in nonunion development and are difficult to diagnose preoperatively due to the lack of clinical signs of infection and unremarkable blood counts. However, our results imply that for low-grade infections, antibiotic therapy may not always be mandatory to heal the nonunion. This study was supported by the German Social Accident Insurance (FF-FR0276)

Aim. Differentiation of infected (INF) nonunion from aseptic (AS) nonunion is crucial for the choice of intra- and postoperative treatment. Preoperative diagnosis of infected nonunion is challenging, especially in case of low-grade infection lacking clinical signs of infection. Standard blood markers such as C-reactive protein or leucocyte count do not aid in preoperative diagnosis. Proteomic profiling has shown promising results for differentiation of numerous chronic disease states, and in this study was applied to preoperative blood samples of patients with nonunion in an attempt to identify potential biomarkers. Method. This prospective multicenter study enrolled patients undergoing revision surgery of femur or tibia nonunion. Patients with implant removal after regular fracture healing (HEAL) were included as a control-group. Preoperative blood samples, intraoperative tissue samples, sonication of osteosynthesis material and 1-year-follow-up questionnaire were taken. Nonunion patients were grouped into INF or AS after assessing bacterial culture and histopathology of retrieved samples. Diagnosis of infection followed the fracture related infection consensus group criteria, with additional consideration of healing one year after revision surgery. Targeted proteomics was used to investigate a predefined panel of 45 cytokines in preoperative blood samples. Statistical differences were calculated with Kruskal Wallis and Dunn's post hoc test. Cytokines with less than 80% of samples being above the lower limit of detection range (LLDR) were excluded for this study. Results. We recruited 62 AS, 43 INF and 32 HEAL patients. Patients in the two nonunion groups (INF and AS) did not differ concerning smoking, diabetes or initial open or closed fracture. Thirty-two cytokines were above LLDR in >80% of patients. INF patients showed a significant difference in expression of 8 cytokines compared to AS, with greatest differences observed for Macrophage Colony Stimulating Factor 1 (MCSF-1) and Hepatocyte Growth Factor (HGF) (p<0.01). In comparing AS with HEAL patients, 9 cytokines displayed significant differences, including interleukin (IL)-6, Vascular Endothelial Growth Factor A (VEGFA), Matrix Metalloproteinase 1 (MMP-1). Comparison of INF with HEAL patients revealed significantly different expression of 20 cytokines, including. IL-6, IL-18, VEGFA or MMP-1. Conclusions. Our study revealed differences in plasma cytokine profile of blood samples from INF and AS patients. Although no single biomarker is sufficient to differentiate these patients preoperatively in isolation, future multivariant analysis of this cytokine data in combination with clinical characteristics may provide valuable diagnostic insights. Funded by German Social Accident Insurance (FF-FR 0276) and AO Trauma (AR2021_04)

The meniscus is at the cornerstone of knee joint function, imparting stability and ensuring shock absorption, load transmission, and stress distribution within the knee joint. However, it is very vulnerable to injury and age-related degeneration. Meniscal tears are reported as the most common pathology of the knee with a mean annual incidence of 66 per 100,000. Knee osteoarthritis progresses more rapidly in the absence of a functional meniscus. Historically, tears extending to the avascular inner portion of the meniscus (white-white zone, “WW”), such as radial tears were considered as untreatable and were often resected, due to the lack of vascularity in the WW zone. Perfusion-based anatomical studies performed on cadaveric menisci in the 1980s shaped the current dogma that human meniscus has poor regenerative capacity, partly due to limited blood supply that only reaches 10 to 25% of the meniscus, commonly referred to as red-red zone (“RR”). Previous studies, including those utilizing animal models have shown mobilization of Mesenchymal Stem Cells (MSCs) upon injury into the WW zone, and successful MSC recruitment when administered externally to the injury site. We and others have recently reported positive outcomes of repaired tears in the inner zone of patients. We hypothesized that the “avascular” white-white zone of the meniscus possesses regenerative capacity due to a resident stem/progenitor cell population. Further, we sought to redefine the presence of microvessels in all meniscal zones using advanced stereology and imaging modalities. Fifteen menisci from fresh human cadaveric knees (mean age: 21.53±6.53 years) without evidence of previous injury were obtained from two tissue banks (JRF, Centennial, CO) and Biosource Medical (Lakeland, FL) and utilized for this study. The use of cadaveric specimens for research purposes was approved by the institutional review board. Tibial plateaus were dissected to harvest medial and lateral menisci along their entire length. The RR, red-white (RW) and WW zones were dissected and separated into three thirds from the inner aspect to the marginal border of the meniscus and their wet weights recorded (Fig.1A). Meniscus tissue cellular content in each zone was obtained from dissociation of meniscus tissue using 0.02% w/v pronase (Millipore) for 1h at 37oC, followed by 18h 0.02% w/v collagenase II (Worthington) at 37oC with shaking. Isolated cells were characterized immediately after harvest using flow cytometry with antibodies against MSCs surface markers (CD105, CD90, CD44 and CD29) as well as respective isotype controls. Further, meniscal cells were cultured and split twice when confluence was reached, characterized at P2 and compared to bone marrow-derived MSCs (BM-MSCs) using the same markers. Self-renewal of cells was assessed using colony forming unit (CFU) assay. Differentiation assays were performed to assess whether colony-forming cells retained multilineage potential. For morphological examination of bigger vessels, samples were fixed in 10% formalin for 1 week, paraffin embedded, sectioned (4 μm thick) and stained with H&E and Masson's trichrome. Presence of microvessels was assessed by CD31 immunofluorescence staining. Further, menisci were cleared using the uDisco protocol labeled with the TO-PRO®-3 stain, a fluorescent dye that stains cell nuclei and imaged using light-sheet microscopy. All continuous data are presented as mean ±standard deviation. Non-repeated measures analysis of variance (ANOVA) and Tukey-Kramer HSD post hoc analysis were performed on sample means for continuous variables. Statistical significance was set at p < 0 .05. Menisci were successfully cleared using a modified uDISCO procedure, imaged and analyzed for total cell density. As expected, bigger vessels were observed in RR but not in WW. However, immunofluorescent staining for CD31 showed a subset of CD31+endothelial cells present in the WW zone, indicating the presence of small vessels, most likely capillaries. In order to assess whether enzymatic digestion had a differential result depending on meniscus zone due to cellular content, we analyzed yields per meniscus per zone. The wet weight of different zones (WW:RW:RR) was at a ratio of ∼1:3:5 respectively, however, the ratio of cells isolated from each zone was at ∼1:4:20, indicating that RR has a denser population of mononuclear cells. However, the difference between all zones in cell yields was not significant. The clonogenic potential of isolated cells was shown to be non-significantly different between the three zones. Differentiation of isolated cells to osteogenic lineage using osteogenic media in vitroshowed no difference between the three zones. Flow cytometry analysis of cells from the three meniscal zones displayed presence of two distinct subpopulations of cells immediately after isolation. One subpopulation was positive to MSC surface markers and the other negative. Additionally, flow cytometry of cultured meniscal cells at P2 displayed that the entire cell population was CD44+CD105+CD29+CD90+, suggesting that culturing meniscal cells results in selection of stem/progenitor cells (plastic adherence). Surface marker expression analysis showed differential expression patterns between markers depending on zone. Similar fraction of cells was detected to express both MSC markers CD90 and CD105 (7–10%) and similar fraction of cells expressed both MSC markers CD29 and CD44 (1–2%) in all three zones, indicating similar density of resident stem/progenitor cells in each zone. Importantly, WW showed significantly higher expression for all four MSC markers compared to the RR zone, indicating higher relative density of stem/progenitor resident cells in the WW zone. Our results determine that CD31-expressing microvessels were present in all zones, including the WW zone, which was previously considered completely avascular. Additionally, stem/progenitor cells were shown to be present in all three zones of the menisci, including the WW zone, showcasing its regenerative potential. For any figures or tables, please contact the authors directly

Although chondrocytes have been used for autologous implantation in defects of articular cartilage, limited availability and donor-site morbidity have led to the search for alternative cell sources. Mesenchymal stem cells from various sources represent one option. The infrapatellar fat-pad is a promising source. Advantages include low morbidity, ease of harvest and ex-vivo evidence of chondrogenesis. Expansion of MSCs from human fat-pad in FGF-2 has been shown to enhance chondrogenesis. To further elucidate this process, we assessed the role of TGF-?3, FGF-2 and oxygen tension on growth kinetics of these cells during expansion. Methods. Infrapatellar fatpads were obtained from 4 donors with osteoarthritis. Cells were expanded in various media formulations (STD, FGF, TGF and FGF/TGF) at both 20% and 5% oxygen tensions. Colony forming unit fibroblast assays were performed for each expansion group and assessed with crystal violet staining. Cell aggregates from each group underwent chondrogenic differentiation in 5% and atmospheric oxygen tension. Pellets were analyzed on day 21. Results. 5% Oxygen tension during expansion increased the colony size for both FGF and FGF/TGF groups. Cells expanded in FGF/TGF proliferated more rapidly. Biochemical analysis revealed that cells expanded in FGF-2 had higher glycosaminoglycan synthesis rates, a marker for chondrogenesis. Differentiation at 5% pO. 2. led to higher levels of sGAG but its effect was generally less potent compared to expansion in FGF-2. Discussion. In agreement with previous findings, expansion of fat-pad MSCs in FGF-2 resulted in enhanced chondrogenesis and increased colony forming capacity. Combined FGF-2 and TGF-?3 during expansion decreased the population doubling time but led to decreased matrix synthesis. Differentiation in low oxygen was beneficial to subsequent chondrogenesis. In conclusion, addition of FGF-2 during the expansion phase was the most potent promoter of the subsequent chondrogenesis of hMSCs isolated from the infrapatellar fat-pad

Introduction. The ability of activated platelets to induce cellular proliferation is well recognised. In a previous diffusion model, platelets combined with Tri-calcium phosphate (TCP) led to an osteoprogenitor mitogenic response followed by cellular differentiation. This study was designed to look at osteoprogenitor responses when cultured directly onto TCP granules combined with activated platelets. Method. Human osteoprogenitors were loaded onto TCP with activated platelets at a low seeding density and high seeding densities. Cellular proliferation was assessed using the pico-green DNA content analysis. Differentiation towards osteoblastic phenotype was assessed using an alkaline phosphatase assay. RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction was used to assess gene expression for type 1 collagen and osteocalcin. Histological assessment for live/dead staining and alkaline phosphatase was used on cultured granule samples. Results. In the low seeding density, platelets induced an early proliferative response compared with controls. After 14 days of culture the cells had not differentiated to an osteoblastic phenotype. When seeded at high densities, cellular differentiation was induced by the activated platelets. Significant cellular proliferation was not observed after seeding at high density. Conclusions. This study demonstrates that osteoprogenitors respond to the local environment which is modulated by both cell-cell contact factors and inflammatory cytokines from the platelets. This study helps to define the concentration of progenitors and platelets needed for further on-growth studies. It may also help define the optimal conditions for seeding cells and platelets for clinical use of composite bone graft substitutes

Purpose. Internal fixation of fractures in the presence of osteopenia has been associated with a failure rate as high as 25%. Enhancing bone formation and osseointegration of orthopaedic hardware is a priority when treating patients with impaired bone regenerative capacity. Fibroblast Growth Factor (FGF) 18 regulates skeletal development and could therefore have applications in implant integration. This study was designed to determine if FGF 18 promotes bone formation and osseointegration in the osteopenic FGFR3−/− mouse and to examine its effect on bone marrow derived mesenchymal stem cells (MSCs). Method. In Vivo: Intramedullary implants were fabricated from 0.4 × 10mm nylon rods coated with 300nm of titanium by physical vapour deposition. Skeletally mature, age matched female FGFR3−/− and wild type mice received bilateral intramedullary femoral implants. Left femurs received an intramedullary injection of 0.1μg of FGF 18 (Merck Serono), and right femurs received saline only. Six weeks later, femurs were harvested, radiographed, scanned by micro CT, and processed for undecalcified for histology. In Vitro: MSCs were harvested from femurs and tibiae of skeletally mature age matched FGFR3−/− and wild type mice. Cells were cultured in Alpha Modified Eagles Medium (αMEM) to monitor proliferation or αMEM supplemented with ascorbic acid and sodium beta-glycerophosphate to monitor differentiation. Proliferation was assessed through cell counts and metabolic activity at days 3, 6 and 9. Differentiation was assessed through staining for osteoblasts and mineral deposition at days 6, 9 and 12. Results. Wild type mice exhibited more peri-implant bone formation compared to FGFR3−/− mice. Peri-implant bone formation at the proximal metaphyseal-diaphyseal junction was increased in FGF18 treated femurs compared with contralateral control femurs in wild type (p = NS) and FGFR3−/− (p = 0.04) mice. Histological analysis corroborated micro CT findings, with FGF 18 treated FGFR3−/− femurs forming peri-implant bone instead of the fibrous response seen in controls. In vitro studies showed that FGF18 significantly increased MSC proliferation and metabolism in a dose dependent manner in wild type and FGFR3−/− mice. Osteoblast differentiation was inhibited by FGF18 in wild type MSCs, but was increased at physiological concentrations in cells harvested from FGFR3−/− mice. Conclusion. FGF 18 increases bone formation and osseointegration of intramedullary implants in osteopenic mice and increases MSC proliferation in both the presence and absence of FGFR3. FGF18 also promoted osteoblast differentiation in the absence of FGFR3 signalling, most likely via FGFR1 or 2. Additional work is needed to confirm the identity of the alternate FGFR and to evaluate its capacity to improve osseous healing in unfavourable in-vivo environments

Aims

The aetiologies of common degenerative spine, hip, and knee pathologies are still not completely understood. Mechanical theories have suggested that those diseases are related to sagittal pelvic morphology and spinopelvic-femoral dynamics. The link between the most widely used parameter for sagittal pelvic morphology, pelvic incidence (PI), and the onset of degenerative lumbar, hip, and knee pathologies has not been studied in a large-scale setting.