The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4). We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes. We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells. We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation. Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._. Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._. This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality

Background. Heterotopic ossification (HO) is lamellar bone formation in the soft tissues following trauma or joint replacement for osteoarthritis (OA). A genome wide association study of HO patients after total hip arthroplasty for OA has identified Kinesin Family Member 26B (KIF26B) as a gene associated with HO severity. KIF26B has previously been associated with HO in mice. Hypothesis and aims: We hypothesised that Kif26b regulates the osteogenic trans-differentiation of myoblasts; a possible mechanism of HO. Using an in vitro model, we wished to establish whether Kif26b is involved in HO formation and to explore the molecular mechanism. Methods. We developed CRISPR/Cas9 mediated Kif26b knockout (KO) C2C12 myoblasts. Wild type (WT) and KO cells were transdifferentiated towards an osteogenic lineage using BMP-2 for 24 days. The effect of Kif26b KO on mineralisation was quantified by calcium staining. The mean difference (±SEM) in gene expression between WT and KO lines was compared with ANOVA. Results. qPCR and western blotting confirmed Kif26b knockout. Kif26b deficient cells produced substantially less mineral versus WT in response to BMP-2 (34.71% ±3.62%, n=12, P<0.0001). At day 8 of osteogenic differentiation, loss of Kif26b abrogated Osterix (113.6 ±6.781 n=5, P<0.0001), Osteocalcin (737.9 ±84.25, n=5, P<0.0001) and Alkaline phosphatase (6989 ±365.7, n=5, P<0.0001) expression, and down regulated Runx2 (2.725 ±0.7724, n=5, P<0.0052) and Collagen type I (7.25 ±1.154, n=5, P<0.0001) expression relative to WT. The knockout cells also appeared morphologically different. Compared to WT, the Kif26b KO cells displayed a less osteoblast-like morphology during transdifferentiation. Conclusion. Our findings demonstrate an undescribed function for Kif26b as a critical regulator of pathological ossification, with a putative role in HO pathogenesis after THA

We report on an innovative surface grafting to highly crosslinked (HXLPE) bearing for THA using a biocompatible-phospholipid-polymer poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC). Such hydrophilic surfaces mimic articular cartilage and are hypothesized to improve lubrication and thereby reduce friction and wear. We performed in vitro testing of wear and friction of ceramic-on-polyethylene THRs with the PMPC treatment, and compared them with untreated controls. Highly cross-linked UHMWPE bearings, gamma-ray-irradiated at different levels with and without vitamin E (HXL Vit. E: 125 kGy, HXL: 75 kGy, respectively) were divided so half were PMPC treated (n=3 for all four groups). All were paired with identical 40 mm diameter zirconia-toughened-alumina ceramic heads. Testing was carried-out on an AMTI hip simulator for 10 million simulated walking cycles with standard lubricant and conditions (ISO-14242-1). Wear was measured gravimetrically at 21 intervals, and so was frictional torque with a previously described and tested methodology. PMPC treatment produced a statistically significant 71% in wear reduction of HXL poly (1.70±1.36 mg/Mc for PMPC vs. 5.86±0.402 mg/Mc for controls, p=0.013). A similar significant wear reduction was found for PMPC treated HXL with Vit. E liners (0.736±0.750 mg/Mc, vs. 2.14±0.269 mg/Mc, p=0.035). The improvements were associated with 12% and 5% reductions in friction of the HXL and Vit. E HXL respectively (statistically significant p=0.003, and marginal p=0.116, one tailed). These results were an important step in the quest for lower wearing, thin and strong UHMWPE liners for larger diameter femoral heads with the potential benefit of longevity and less risk of dislocation after surgery

Aims

This study aimed to determine if macrophages can attach and directly affect the oxide layers of 316L stainless steel, titanium alloy (Ti6Al4V), and cobalt-chromium-molybdenum alloy (CoCrMo) by releasing components of these alloys.

Introduction

Metal alloys have been commonly used for surgical applications due to their suitable mechanical characteristics and relatively good biocompatibility. However, direct cellular corrosion of orthopaedic implants remains a controversial topic and is still not fully understood. This study aims to examine a possible aspect of this corrosion mechanism by determining if macrophages can attach and directly affect the surfaces of 316L stainless steel, Ti6Al4V, and CoCrMo by releasing components of the alloy oxide layer.

Although there is some clinical evidence of ceramic bearings being associated with a lower infection rate after total hip arthroplasty (THA), available data remains controversial since this surface is usually reserved for young, healthy patients. Therefore, we investigated the influence of five commonly-used biomaterials on the adhesion potential of four biofilm-producing bacteria usually detected in infected THAs.

In this in-vitro research, we evaluated the ability of S. aureus, S. epidermidis ATCC 35984, E. coli ATCC 25922 and P. aeruginosa to adhere to the surface of solid biomaterials, including a 28mm cobalt-chromium metal head, a 28mm fourth-generation ceramic head, a 48mm fourth-generation ceramic insert, a 48mm highly-crossed linked polyethylene insert and a 52mm titanium porous-coated acetabular component. After an initial vortex step, a bacterial separation from the surface of each specimen was done until no remaining attached bacteria were observed by digital optical microscope. The colony-forming units were counted to determine the number of viable adherent bacteria and the bacterial density.

We found no differences on global bacterial adhesion between the different surfaces. E. coli presented the least adherence potential among the analysed pathogens (p<0.001). The combination of E. coli and S. epidermidis generated an antagonist effect over the adherence potential of S. epidermidis individually (58±4% vs. 48±5%; p=0.007). The combination of P. aeruginosa and S. aureus presented a trend to an increased adherence of P. aeruginosa independently, suggesting an agonist effect (71% vs. 62%; p=0.07).

In this study, ceramic bearings appeared not to be related to a lower bacterial adhesion than other biomaterials. However, different adhesive potentials among bacteria may play a major role on infection's inception.

Introduction

Recent studies on large diameter femoral head hip replacements have implicated the modular taper junction as one of the significant sources of wear and corrosion products and this has been attributed to increased torque and bending on the taper interface. The aim of this study was to assess the effect of frictional torque and bending moment on fretting corrosion at the taper junction and to investigate whether different material combinations also had an effect.

Aims. To evaluate the effect of ultrasound-targeted simvastatin-loaded microbubble destruction (UTMDSV) for alleviation of the progression of osteoarthritis (OA) in rabbits through modulation of the peroxisome proliferator-activated receptor (PPARγ). Methods. In vitro, OA chondrocytes were treated with ultrasound (US), US-targeted microbubble destruction (UTMD), simvastatin (SV), and UTMDSV on alternate days for four weeks. Chondrocytes were also treated with PPARγ inhibitor, PPARγ inhibitor+ UTMDSV, and UTMDSV. The cholesterol efflux rate and triglyceride levels were measured using an assay kit and oil red O staining, respectively. In vivo, the OA rabbits were treated with a single intra-articular injection of UTMD, SV, and UTMDSV every seven days for four weeks. Cartilage histopathology was assessed by safranin-O staining and the Mankin score. Total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) in rabbit knee synovial fluid were detected by enzyme-marker assay. Aggrecan, collagen II, and PPARγ expression levels were analyzed by Western blotting (WB). Results. In vitro, UTMDSV significantly increased the cholesterol efflux rate and aggrecan, collagen II, and PPARγ levels in OA chondrocytes; these effects were blocked by the PPARγ inhibitor. In vivo, UTMD. SV. significantly increased aggrecan, collagen II, PPARγ, and HDL-C levels, while TC levels and Mankin scores were decreased compared with the UTMD, SV, OA, and control groups. Conclusion. UTMDSV promotes cartilage extracellular matrix synthesis by modulating the PPARγ-mediated cholesterol efflux pathway in OA rabbits. Cite this article: Bone Joint Res 2021;10(10):693–703

Aims. Current treatments of prosthetic joint infection (PJI) are minimally effective against Staphylococcus aureus biofilm. A murine PJI model of debridement, antibiotics, and implant retention (DAIR) was used to test the hypothesis that PlySs2, a bacteriophage-derived lysin, can target S. aureus biofilm and address the unique challenges presented in this periprosthetic environment. Methods. The ability of PlySs2 and vancomycin to kill biofilm and colony-forming units (CFUs) on orthopaedic implants were compared using in vitro models. An in vivo murine PJI model of DAIR was used to assess the efficacy of a combination of PlySs2 and vancomycin on periprosthetic bacterial load. Results. PlySs2 treatment reduced 99% more CFUs and 75% more biofilm compared with vancomycin in vitro. A combination of PlySs2 and vancomycin in vivo reduced the number of CFUs on the surface of implants by 92% and in the periprosthetic tissue by 88%. Conclusion. PlySs2 lysin was able to reduce biofilm, target planktonic bacteria, and work synergistically with vancomycin in our in vitro models. A combination of PlySs2 and vancomycin also reduced bacterial load in periprosthetic tissue and on the surface of implants in a murine model of DAIR treatment for established PJI. Cite this article: Bone Joint J 2020;102-B(7 Supple B):3–10

Aims. The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells. Methods. HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 10. 6. PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%. Conclusion. The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF. Cite this article: Bone Jt Open 2022;3(4):340–347

Polymethylmethacrylate (PMMA) bone cement is strong in compression, however it tends to fail under torsion. Sufficient pressurisation and subsequent interdigitation between cement and bone are critical for the mechanical interlock of cemented orthopaedic implants, and an irregular surface on the acetabular cup is necessary for reasonable fixation at the cup-cement interface. There is limited literature investigating discrepancies in the failure mechanisms of cemented all-polyethylene acetabular cups with and without cement spacers, under torsional loading. In vitro experimental comparison of three groups of polyethylene acetabular prosthesis (PAP) cemented into prepared sawbone hemipelvises:. * PAP without PMMA spacers maintaining an equal cement mantle circumferentially. (Group 1 n=3). * PAP without PMMA spacers cemented deliberately ‘bottoming-out’ the implant within the acetabulum. (Group 2 n=3). * PAP with PMMA spacers. (Group 3 n=3). The constructs were tested to torstional failure on a custom designed setup, and statistical analysis done by a one-way ANOVA and Tukey-Welsh test. Group 3 demonstrated superior torsional resistance with a statistically significant torque of 145Nm (SD±12Nm) at failure, compared to group 2 (109Nm, SD±7Nm) and group 1 (99Nm, SD±8Nm). Group 3 experienced failure predominantly at the bone-cement interface, in contrast, Groups 1 and 2 exhibited failure predominantly at the cup-cement interface. There was no significant difference between Group 1 and 2. Qualitative analysis of the failure mode indicates the efficient redistribution of stress throughout the cement mantle, consistent with the greater uniformity of cement. PMMA spacers increase the resistance to torsional failure at the implant-cement interface. Acetabular components without spacers (Groups 1 and 2) failed at the implant-cement interface before the cement-bone interface, at a statistically significantly lower level of torque to failure. Although the PMMA spacers may reduce cement interdigitation at the cement-bone interface the torsional forces required to fail are likely supraphysiological

Bone marrow stem cells (BMSCs) represent a collection of different cell types exhibiting stem cell characteristics but with notable heterogeneity. Among these, Skeletal Stem Cells (SSCs) represent a distinct matrix subgroup within BMSC and demonstrate a specialized capacity to facilitate bone formation, recruit chondrocytes, and contribute to hematopoiesis. SSCs play a pivotal role in orchestrating the functions of skeletal organs. Local ischemia has a significant impact on cell survival and function. We hypothesize that bone ischemia induces alterations in the differentiation potential of SSCs, consequently influencing changes in bone structure. We mechanically dissected tissue from the necrotic segment in the femoral head and more normal appearing areas from the femoral neck of specimens from 5 patients diagnosed with osteonecrosis of the femoral head (ONFH). These tissues were enzymatically broken down into individual cell suspensions. Utilizing fluorescence-activated cell sorting (FACS) based on specific surface markers indicative of human skeletal stem cells (hSSC), namely CD45- CD235a- CD31- TIE2- Podoplanin (PDPN)+ CD146- CD73+ CD164+, we isolated a distinct cell population. Subsequent in vitro evaluations, focusing on clonogenicity, osteogenesis, and chondrogenesis were conducted to assess the functional prowess of these SSCs. Moreover, we introduced BMP2 at a concentration of 50ng/ml to SSCs extracted from necrotic regions to potentially reinstate their osteogenic capabilities. We effectively isolated SSCs from both Necrotic and Non-necrotic Zones. We observed an augmented clonal formation capacity and chondrogenesis ability of SSCs isolated from the necrotic region, accompanied by a significant decline in osteogenic ability (P＜0.01), an effect not reversible even with the addition of BMP2. Ischemia adversely affects the proliferation and function of SSCs, resulting in a diminished osteogenic capacity and an insensitivity to BMP2, ultimately leading to structural alterations in bone tissue

Aims. This combined clinical and in vitro study aimed to determine the incidence of liner malseating in modular dual mobility (MDM) constructs in primary total hip arthroplasties (THAs) from a large volume arthroplasty centre, and determine whether malseating increases the potential for fretting and corrosion at the modular metal interface in malseated MDM constructs using a simulated corrosion chamber. Methods. For the clinical arm of the study, observers independently reviewed postoperative radiographs of 551 primary THAs using MDM constructs from a single manufacturer over a three-year period, to identify the incidence of MDM liner-shell malseating. Multivariable logistic regression analysis was performed to identify risk factors including age, sex, body mass index (BMI), cup design, cup size, and the MDM case volume of the surgeon. For the in vitro arm, six pristine MDM implants with cobalt-chrome liners were tested in a simulated corrosion chamber. Three were well-seated and three were malseated with 6° of canting. The liner-shell couples underwent cyclic loading of increasing magnitudes. Fretting current was measured throughout testing and the onset of fretting load was determined by analyzing the increase in average current. Results. The radiological review identified that 32 of 551 MDM liners (5.8%) were malseated. Malseating was noted in all of the three different cup designs. The incidence of malseating was significantly higher in low-volume MDM surgeons than high-volume MDM surgeons (p < 0.001). Pristine well-seated liners showed significantly lower fretting current values at all peak loads greater than 800 N (p < 0.044). Malseated liner-shell couples had lower fretting onset loads at 2,400 N. Conclusion. MDM malseating remains an issue that can occur in at least one in 20 patients at a high-volume arthroplasty centre. The onset of fretting and increased fretting current throughout loading cycles suggests susceptibility to corrosion when this occurs. These results support the hypothesis that malseated liners may be at risk for fretting corrosion. Clinicians should be aware of this phenomenon. Cite this article: Bone Joint J 2020;102-B(7 Supple B):20–26

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570

Aims. The aim of this study was to estimate the 90-day risk of revision for periprosthetic femoral fracture associated with design features of cementless femoral stems, and to investigate the effect of a collar on this risk using a biomechanical in vitro model. Materials and Methods. A total of 337 647 primary total hip arthroplasties (THAs) from the United Kingdom National Joint Registry (NJR) were included in a multivariable survival and regression analysis to identify the adjusted hazard of revision for periprosthetic fracture following primary THA using a cementless stem. The effect of a collar in cementless THA on this risk was evaluated in an in vitro model using paired fresh frozen cadaveric femora. Results. The prevalence of early revision for periprosthetic fracture was 0.34% (1180/337 647) and 44.0% (520/1180) occurred within 90 days of surgery. Implant risk factors included: collarless stem, non-grit-blasted finish, and triple-tapered design. In the in vitro model, a medial calcar collar consistently improved the stability and resistance to fracture. Conclusion. Analysis of features of stem design in registry data is a useful method of identifying implant characteristics that affect the risk of early periprosthetic fracture around a cementless femoral stem. A collar on the calcar reduced the risk of an early periprosthetic fracture and this was confirmed by biomechanical testing. This approach may be useful in the analysis of other uncommon modes of failure after THA. Cite this article: Bone Joint J 2019;101-B:779–786

Objectives. Studies reporting specifically on squeaking in total hip arthroplasty have focused on cementless, and not on hybrid, fixation. We hypothesised that the cement mantle of the femur might have a damping effect on the sound transmitted through the metal stem. The objective of this study was to test the effect of cement on sound propagation along different stem designs and under different fixation conditions. Methods. An in vitro model for sound detection, composed of a mechanical suspension structure and a sound-registering electronic assembly, was designed. A pulse of sound in the audible range was propagated along bare stems and stems implanted in cadaveric bone femurs with and without cement. Two stems of different alloy and geometry were compared. Results. The magnitudes of the maximum amplitudes of the bare stem were in the range of 10.8 V to 11.8 V, whereas the amplitudes for the same stems with a cement mantle in a cadaveric bone decreased to 0.3 V to 0.7 V, implying a pulse-attenuation efficiency of greater than 97%. The same magnitude is close to 40% when the comparison is made against stems implanted in cadaveric bone femurs without cement. Conclusion. The in vitro model presented here has shown that the cement had a remarkable effect on sound attenuation and a strong energy absorption in cement mantle and bone. The visco-elastic properties of cement can contribute to the dissipation of vibro-acoustic energy, thus preventing hip prostheses from squeaking. This could explain, at least in part, the lack of reports of squeaking when hybrid fixation is used. Cite this article: F. J. Burgo, D. E. Mengelle, A. Ozols, C. Fernandez, C. M. Autorino. The damping effect of cement as a potential mitigation factor of squeaking in ceramic-on-ceramic total hip arthroplasty. Bone Joint Res 2016;5:531–537. DOI: 10.1302/2046-3758.511.BJR-2016-0058.R1

Aims

Several short- and mid-term studies have shown minimal liner wear of highly cross-linked polyethylene (HXLPE) in total hip arthroplasty (THA), but the safety of using thinner HXLPE liners to maximize femoral head size remains uncertain. The objective of this study was to analyze clinical survival and radiological wear rates of patients with HXLPE liners, a 36 mm femoral head, and a small acetabular component with a minimum of ten years’ follow-up.

Aims

Limited implant survival due to aseptic cup loosening is most commonly responsible for revision total hip arthroplasty (THA). Advances in implant designs and materials have been crucial in addressing those challenges. Vitamin E-infused highly cross-linked polyethylene (VEPE) promises strong wear resistance, high oxidative stability, and superior mechanical strength. Although VEPE monoblock cups have shown good mid-term performance and excellent wear patterns, long-term results remain unclear. This study evaluated migration and wear patterns and clinical and radiological outcomes at a minimum of ten years’ follow-up.

Aims

Iliopsoas impingement occurs in 4% to 30% of patients after undergoing total hip arthroplasty (THA). Despite a relatively high incidence, there are few attempts at modelling impingement between the iliopsoas and acetabular component, and no attempts at modelling this in a representative cohort of subjects. The purpose of this study was to develop a novel computational model for quantifying the impingement between the iliopsoas and acetabular component and validate its utility in a case-controlled investigation.

Aims

The aim of the study was to investigate whether the primary stability of press-fit acetabular components can be improved by altering the impaction procedure.