Aims

Our purpose was to describe an unusual series of 21 patients with fungal osteomyelitis after an anterior cruciate ligament reconstruction (ACL-R).

Osteoinductive bone substitutes are in their developmental infancy and a paucity of effective grafts options persists despite clinical demand. Bone mineral substitutes such as hydroxyapatite cause minimal biological activity when compared to osteoinductive systems present biological growth factors in order to drive bone regeneration. We have previously demonstrated the in-vitro efficacy of a bioengineered system at presenting growth factors at ultra low-doses. This study aimed to translate this growth factor delivery system towards a clinically applicable implant.

Osteoinductive surfaces were engineered using plasma polymerisation of poly(ethyl acrylate) onto base materials followed by adsorption of fibronectin protein and subsequently growth factor (BMP-2). Biological activity following ethylene oxide (EO) sterilisation was evaluated using ELISAs targeted against BMP-2, cell differentiation studies and atomic force microscopy. Scaffolds were 3D printed using polycaprolactone/hydroxyapatite composites and mechanically tested using a linear compression models to calculate stress/strain. In-vivo analysis was performed using a critical defect model in 23 mice over an 8 week period. Bone formation was assessed using microCT and histological analysis. Finally, a computer modelling process was developed to convert patient CT images into surface models, then formatted into 3D-printable scaffolds to fill critical defects.

Following EO sterilisation, there was no change in scaffold surface and persistent availability of growth factors. Scaffolds showed adequate porosity for cell migration with mechanical stiffness similar to cancellous bone. Finally, the in vivo murine model demonstrated rapid bone formation with evidence of trabecular remodelling in samples presenting growth factors compared to controls.

Aim

Late acute prosthetic joint infections (PJI) treated with surgical debridement and implant retention (DAIR) have a high failure rate. The aim of our study was to evaluate treatment outcome in late acute PJIs treated with DAIR versus implant removal.

Aim

Debridement, antibiotics and implant retention (DAIR) is the recommended treatment for all acute prosthetic joint infections (PJI). However, the efficacy of DAIR and identification of risk factors for failure in patients with late acute PJI, is not well described.

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Existing regenerative approaches rely on high doses of growth factors, such as BMP-2 in bone regeneration, which can cause serious side effects. We describe an ultra-low-dose growth factor technology yielding high bioactivity based on a simple polymer, poly (ethyl acrylate) (PEA), and report its translation to a clinical veterinary setting. This polymer-based technology triggers spontaneous fibronectin organization and stimulates growth factor signaling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, we use plasma-polymerized PEA on 2D and 3D substrates to enhance cell signaling in vitro, showing the complete healing of a critical-size bone injury in mice in vivo. We demonstrate its safety and efficacy in a Münsterländer dog with a non-healing humerus fracture, establishing the clinical translation of advanced ultra-low-dose growth factor treatment.

Material-based strategies seek to engineer synthetic microenvironments that mimic the characteristics of physiological extracellular matrices for applications in regenerative therapies, including bone repair and regeneration. In our group, we identified a specific chemistry, poly(ethyl acrylate) (PEA), able to induce the organization of fibronectin (FN), upon adsorption of the protein, into fibrillar networks similar to the physiological ones, leading to enhanced cellular response, in terms of cell adhesion and differentiation. In this work, we exploit these FN networks to capture and present growth factors (GF) in combination with the integrin binding domain of FN during bone tissue healing.

Fibrillar conformation of FN adsorbed on PEA favors the simultaneous availability of the GF binding domain (FNIII12–14) next to the integrin binding region (FNIII9–10), compared to poly(methyl acrylate) (PMA), a material with similar chemistry, where FN adopts a globular conformation. The combined exposure of specific adhesive sequences recognized by integrins and GF binding domains was found to improve the osteogenic differentiation of mesenchymal stem cells. A higher expression of bone proteins was found when BMP2 is bound or sequestered on the material surface versus its administration in the culture media in vitro. The potential of this system as recruiter of GFs was also investigated in a critical-size bone segmental defect in mouse. The synergistic integrin-GF signalling, induced by fibrillar FN, promoted bone formation in vivo with lower BMP2 doses than current technologies. Furthermore, we optimized the system for its potential use in translational research, seeking to address the clinical need of using biocompatible and biodegradable material implants. Polycaprolactone scaffolds were synthesized and coated with a thin layer of plasma- polymerized PEA that recruits and efficiently presents GF during healing of critical size defects.

The material-driven FN fibrillogenesis provides a new strategy to efficiently reduce the GF doses administrated in bone regenerative therapies.

Polyether ether ketone (PEEK) has been increasingly employed as biomaterials for trauma, orthopeadic, and spinal implants. However, concern has been raised about the inertness of PEEK which limits bone integration. In this study, we have coated PEEK with a functional material seeking to promote osteogenic differentiation of human mesenchymal stem cells (hMSC).

We have used spray drying to coat poly(ethyl acrylate) (PEA) as a coating on PEEK. This technique is simple, allows a range of controlled coating thicknesses (from hundred nm to a few um), cost effective and easily translatable to scaffolds or implant surfaces for existing or new orthopaedic applications. PEA induces the organisation of fibronectin (FN) into nanonetworks upon simple adsorption from protein solutions. These FN nanonetworks on PEA represent a microenvironment for efficient growth factor binding and presentation in very low but effective doses. In this study we show cell adhesion and stem cell differentiation towards an osteogenic lineages when bone morphogenetic protein 2 (BMP2) was adsorbed on these engineered PEEK/PEA/FN microenvironments in very low doses.

Overall, the developed functional coatings on PEEK has the potential to allow the translation of this material into orthopaedic applications.

Control of stem cell fate and function is critical for clinical and academic work. By combining surface chemistry-driven extracellular matrix (ECM) assembly with mesenchymal stem cells (MSCs) we are developing a system which can be used to regulate the behaviour of MSCs. The conformation of the ECM glycoprotein fibronectin (Fn) is different when adsorbed onto poly methylacrylate (PMA) where it is globular, and on poly ethylacrylate (PEA) where it forms a physiologically-similar network^[1] (Fig. 1). Using these polymers to govern Fn conformation, we are developing a 3D system incorporating MSC-responsive growth factors (GFs) and bone marrow MSCs capable of regulating MSC behaviour.

Toluene-dissolved PMA and PEA were spin coated onto glass coverslips before solvent extraction in vacuo and UV sterilisation. 20 mg ml⁻¹ human plasma FN was adsorbed onto the surfaces followed by 25 ng ml⁻¹ recombinant human BMP2/VEGF. FN conformations were characterised by atomic force microscopy (AFM). A collagen hydrogel was placed above the substrate. Adult human bone marrow STRO-1+ were cultured on the substrates for 3 weeks in supplemented DMEM. Expression of MSC stemness and HSC maintenance factors were analysed by In-Cell Western assay.

To establish the best combination of polymer/FN/GF, MSC stemness markers (ALCAM, NESTIN and STRO1), osteogenic differentiation markers (OCN and OPN) and bone marrow markers (SCF and VCAM1) were measured in MSCs cultured for 3-weeks on substrates. OCN, SCF, and VCAM1 expression was enhanced across all combinations compared to glass control, while for ALCAM/STRO1/NESTIN and OPN, PEA combinations enhanced their expression. PEA + FN + VEGF appeared to be system best suited to maintaining MSC stemness and supporting expression of osteogenesis markers and bone marrow markers.

We have shown that MSCs maintain their stem cells state and express high levels of SCF and VCAM-1 when cultured on PEA with adsorbed Fn and VEGF or BMP2. Next stages of this work will use PCR to verify results and analyse expression of other MSC markers to develop a role for these synthetic polymers as biomaterials.

Objectives: To explore the potential clinical benefits of PRGF injections for the treatment of OA in a retrospective observational study and to characterize the PRGF treatment in OA patients.

Methods: A total of 62 patients with symptomatic OA (Knee OA, 41 patients; hip OA, 21 patients) were treated with a series of three weekly intra-articular injections of PRGF and studied retrospectively. ELISA assays were used to determine the levels of VEGF-A, HGF, PDGF, TGF-β1 and IGF-I in PRGF. The patients completed the WOMAC questionnaires prior to PRGF treatment at two and six months after its instauration. The primary efficacy criteria were mean change from baseline through two and six months in the WOMAC index pain and physical function scores. Change scores for the Harris hip scores were calculated for 6 months post-treatment. Age and BMI were included in the models.

results The mean age and BMI of the participants with hip and knee OA were 59 and 60 years and 27.8 and 28.5 kg/m2 respectively. In knee OA the differences between pain scores at baseline and two or six months were highly significant (−1.766, 95% CI: −1.073 to −2.458, p=0.000, and −2.320, CI: −3.838 to −0.803, p=0.011) The observed success rates for the pain sub-scale reached 37% by two months and 31.7% by six months. After two months, WOMAC physical function scores decreased significantly (−4.772, 95% CI: −6.864 to −2.681, p=0.000). The changes at six months were not statistically significant (n=41). The success rates for the physical function subscale were 31.4% by two months and 31.7% by six months (n=41). In hip OA the differences between WOMAC pain and Harris hip core scores at baseline and six months were significant. The success rate for the pain subscale and Harris hip score reached 58% and 85% by six months

PRGF resulted in a moderate enrichment in platelet number, 2.0 ± 0.5-fold increase compared to peripheral blood. The levels of the main platelet secretory growth factors were 27.28 ± 10.90 ng/cc for TGF-β1 and 15.66 ± 8.02 ng/cc for PDGF. VEGF was also secreted from platelets but was less abundant, 437± 446 pg/cc. Other GFs present in PRGF refiect mainly plasma levels; among these growth factors are IGF-I (55.53 ± 20.87 ng/cc) and the less concentrated HGF (472 ± 221 pg/cc).

Discussion: Due to the localized nature of OA, the possibility of intra-articular administration of PRGF, along with its biocompatibility and non-immunogenicity, may make this unique molecular mixture an attractive treatment for OA. PRGF may have therapeutic effects in OA joints via multiple biologic mechanisms. The results of this study will give a first impression of potential effectiveness of PRGF for the local treatment of hip and knee OA.

35 patients with an infected total knee arthroplasty were operated with a two-stage revision protocol including the use of custom hand-made antibiotic loaded articulating spacers.

Spacers were built intraoperatively, without specific tools, regarless the defect being considered cavitary or segmentary.

Patients were allowed to walk with an orthosis. Range of motion (ROM) with the articulating spacer averaged 80° and after reimplantation 106.5°. All but two patients in our series were treated with a combination of antibiotics including rifampicin and the antibiotics used in the spacers constituted from 7.5% of its final weight.

Reimplantation was successfully performed in 33 out of 35 cases at an average time of 10.2 weeks, excluding a patient were we had to wait 2.5 years. An extended exposure at reimplantation was necessary in 21% of the patients (five “Q-snip” and two anterior tibial tuberosity osteotomies).

According to the Knee Society Score (KSS) the results were considered excellent or good in 27 patients (84.8%), and fair or poor in 6 patients (14.2%) out of the 33 reimplantated. No significant differences related to the micro-organism or the time elapsed until reimplantation, as compared for ROM and functional and clinical KSS were found but early infections had significant worst ROM than late or sub-acute infections. Intercondylar constrained designs had better functional, clinic KSS and ROM that posterostabilised designs or hinge designs without significant differences.

Our modified technique for custom made spacers can be applied in any surgical theatre with a minimum cost. Our results are comparable to those reported in the literature, demonstrating the consistency of the two-stage reimplantation protocol despite multiple modifications and different dosages of antibiotic used in the cement spacers.