Our previous rat study demonstrated an ex vivo-created “Biomimetic Hematoma” (BH) that mimics the intrinsic structural properties of normal fracture hematoma, consistently and efficiently enhanced the healing of large bone defects at extremely low doses of rhBMP-2 (0.33 μg). The aim of this study was to determine if an extremely low dose of rhBMP-2 delivered within BH can efficiently heal large bone defects in goats. Goat 2.5 cm tibial defects were stabilized with circular fixators, and divided into groups (n=2-3): 2.1 mg rhBMP-2 delivered on an absorbable collagen sponge (ACS); 52.5 μg rhBMP-2 delivered within BH; and an empty group. BH was created using autologous blood with a mixture of calcium and thrombin at specific concentrations. Healing was monitored with X-rays. After 8 weeks, femurs were assessed using microCT. Using 2.1 mg on ACS was sufficient to heal 2.5 cm bone defects. Empty defects resulted in a nonunion after 8 weeks. Radiographic evaluation showed earlier and more robust callus formation with 97.5 % (52.5 μg) less of rhBMP-2 delivered within the BH, and all tibias were fully bridged at 3 weeks. The bone mineral density was significantly higher in defects treated with BH than with ACS. Defects in the BH group had smaller amounts of intramedullary and cortical trabeculation compared to the ACS group, indicating advanced remodeling. The results confirm that the delivery of rhBMP-2 within the BH was much more efficient than on an ACS. Not only did the large bone defects heal consistently with a 40x lower dose of rhBMP-2, but the quality of the defect regeneration was also superior in the BH group. These findings should significantly influence how rhBMP-2 is delivered clinically to maximize the regenerative capacity of bone healing while minimizing the dose required, thereby reducing the risk of adverse effects

Background. Tranexamic acid (TXA) and fibrin sealants have gained widespread use in total knee arthroplasty. They can decrease bleeding, drainage volume, hematoma formation, and incidence of blood transfusion. However, they are costly and carry a theoretical risk of infection transmission and thrombosis. This study compares the two pharmacologic interventions to preoperative autologous blood donation as well as no intervention. Methods. This prospective study evaluated a process change within our blood management program over the last five years. The program began initially with a comparison of only routine hemostasis compared to routine preoperative autologous blood donation (PABD) for all patients (Group 1), which then evolved into a targeted PABD protocol where only anaemic patients predonated (Group 2). Subsequently, patients received topical fibrin sealant for a year (Group 3), after which the topical TXA protocol was introduced and is still in place (Group 4). Results. 838 patients went through the blood management protocols. 86 patients (10%) received allogeneic blood: 6 (5%) in Group 1; 18 (9%) in Group 2; 18 (14%) in Group 3; 20 (7%) in Group 4, and 24 (26%) in the control group. No significant difference was observed between the fibrin sealant group and the TXA group with regard to the need for transfusion, but both were significantly lower than controls. The TXA group registered the lowest volume of blood loss, shortest length of stay, and lowest cost. These results were more pronounced in anaemic patients. Conclusion. Both TXA and fibrin sealants were effective in reducing transfusion risk compared to control as well as PABD in primary unilateral total knee arthroplasty. Given the equivalent eαects of both pharmacologic interventions in this study, together with the cost considerations and theoretical harms from the use of blood-derived products, it would seem prudent to use tranexamic acid in preference to fibrin sealants

Chronic Achilles tendinopathy is characterised by sub-acute inflammation with pro-inflammatory type 1 macrophages (M1), tissue degeneration and consequent partial or total tendon injury. Control of the inflammatory response and M1-to-M2 macrophage polarisation can favour tendon healing both directly and indirectly, by allowing for the regenerative process driven by local mesenchymal stem cells. Ten patients (3 females and 7 males aged between 32 and 71 years old) with partial Achilles tendon injury were treated with injections of autologous peripheral blood mononuclear cells (PB-MNCs). The cell concentrate was obtained from 100-120 cc of each patient's blood with a selective point-of-care filtration system. PB-MNCs remained trapped in the filter and were injected immediately after sampling. Around 60% of the PB-MNC concentrate was injected directly into the injured area, while the remaining 40% was injected in smaller amounts into the surrounding parts of the Achilles tendon affected by tendinosis. All patients were evaluated both clinically with the help of the American Orthopaedic Foot & Ankle Society (AOFAS) scale, and radiologically (MRI examination) at baseline and 2 months after the PB-MNC injection. A clinical reassessment with the AOFAS scale was also performed 6 months after the intervention. The rehabilitation protocol implied full weight-bearing walking immediately after the procedure, light physical activity 3-4 days after the injection, and physiotherapist-assisted stretching exercises and eccentric training. In all patients, functional and radiological signs of tendon healing processes were detected as early as 2 months after a single treatment and the AOFAS scale rose from the initial mean value of 37.5 (baseline) to 85.4 (6 months). Our preliminary results indicate that regenerative therapies with PB-MNCs can prove useful for partial Achilles tendon injuries as a valid alternative to surgical options, especially when other conservative approaches have failed. Advantages of this therapy include rapid execution, no need for an operating theatre, easy reproducibility, quick recovery and good tolerability regardless of the patient's age (the procedure is not to be performed in subjects who are below 18 years old). Further studies on the topic are recommended to confirm these observations

Blood management protocols attempt to reduce blood loss by strategies including autologous blood donation, red cell salvage, normovolaemic haemodilution and haemostatic agents such as tranexamic acid (TXA). TXA usage in particular has become increasingly commonplace with numerous studies demonstrating a significant reduction in peri-operative blood loss and proportion of patients requiring transfusion, without increasing the risk of venous thromboembolism. Tourniquet usage has now become ubiquitous in TKA operations with reported benefits of improved visualization, shorter operative time and decreased intra-operative bleeding. However, its use is not without considerable complications including wounding dehiscence, increased venous thromboembolism, superficial wound infection and skin blistering. It is therefore imperative that we review tourniquet usage in light of ever evolving blood management strategies. The aim of this study was to evaluate the effect of stopping tourniquet usage in primary TKRs, performed by an experienced surgeon, in light of new blood reduction measures, such as a TXA. A retrospective analysis identified a total of 31 patients who underwent primary TKR without the use of a tourniquet from January 2018 to March 2019. This was compared to an earlier group of patients from the same surgeon undergoing TKR with the use of a tourniquet; dating from July 2016 to November 2017. All surgeries were performed within the same hospital (CXH). Peri-operative factors and outcome measures were collected for analysis. There was no significant difference in post-operative haemoglobin drop (Tourniquet, 23.1 g/L; No Tourniquet, 24.4 g/: p=0.604) and fall in haematocrit (Tourniquet, 0.082; No Tourniquet, 0.087: p=0.604). Allogenic blood transfusion rates were the same in both groups at 12.9% (2 patients) and blood loss was not found to be significantly different (Tourniquet, 1067ml; No tourniquet, 1058mls). No significant difference was found in operative time (Tourniquet, 103 minutes; No Tourniquet, 111.7 minutes: p=0.152) or length of stay (Tourniquet, 5.5 days; No Tourniquet, 5.2 days: p=0.516). Tranexamic acid usage was not found to be significant (p=1.000). ROM of motion and analgesia requirement was significantly better in the no tourniquet group on one post-operative day out of five analysed (p=0.025, p=00.011). No post-operative thromboembolic events were reported in either group. There was no significant difference in readmission rates (p=0.492) or complications (p=0.238). The increase in minor complications and potential increased VTE risk with tourniquet usage must be balanced against an improved visual field and reduced blood loss in TKR patients. Our study found no difference in post-operative blood loss and transfusion rates between tourniquet and no tourniquet groups. With ever evolving and improving blood loss management strategies, including the use of TXA, the application of tourniquet may not be needed. Further prospective RCTs are needed to assess the impact of tourniquet usage in light of this

Angiogenesis is a key factor in early stages of wound healing and is crucial for tissue regeneration. Gold standard for large bone defect treatment is the transplantation of autologous bone grafts, but is not entirely satisfying (e.g. limited amount). Cell therapies and tissue engineering approaches may overcome these problems by using cells and autologous blood components obtainable by less invasive procedures. Pre-clinical studies previously showed promising results combining endothelial progenitor cells (EPCs) and mesenchymal stem cell (MSCs) in polyurethane scaffolds in presence of PRP (1). A systemic investigation of the chemical and mechanical characteristics of different PRP gels formulations suggested their potential use as sustained autologous growth factor delivery system (2). Here we investigate PRP hydrogels as autologous injectable cell delivery systems for EPCs and MSCs and their efficacy in promoting fast neo-vascularization for bone repair applications. PRP hydrogel and corresponding platelet lysate (PL) were produced from platelet concentrates as described before (3). MSCs were isolated by Ficoll-Paque centrifugation from human bone marrow (EK_regensburg12-101-0127), and cultured in alpha MEM containing 10% FCS and 5 ng/mL basic-FGF (GIBCO). EPCs (CD133+/CD34+) were isolated from MSC fractions using magnetic-activated cell sorting (MACS®) and further cultured in IMDM (GIBCO) containing 5% FCS and 5% PL. GFP positive HUVECs are from Angio-Proteomie, (Boston, USA). Prior to gel encapsulation, MSC and EPCs were pre-stained using PKH26-red® and PKH67-green® respectively. Cells in different proportions were encapsulated in 3D PRP gels, in FDA approved Fibrin gels and in Matrigel®. The gels were cultured in Ibidi microwells placed in an onstage incubator linked to an EVOS Auto Cell Imaging System. The cellular network formation capacity of HUVEC or EPCs and MSC in different proportions was analyzed for the 3 types of hydrogels using time lapse movies recorded over a period of 14 days. Parallel cultures were performed in a classical cell culture CO. 2. incubator and sample gels were taken at different time points for additional immunostaining and gene expression analysis. Preliminary results indicate high cell viability in all of the three tested gels. PRP hydrogels present a favorable environment for the formation of a 3 dimensional cellular network in cell co-culture. The formation of these networks was apparent as early as 4 days after seeding. Networks increase in complexity and branching over time. The same was observed when cells were embedded in Matrigel®, which is known for its pro-angiogenic properties. Further experiments are currently in process looking at the involvement of MSCs in this process and the effect of PRP 3D co-culture on their differentiation. PRP was previously shown as a potent growth factor delivery system for tissue engineering. In the present work, the high cell viability together with the 3 dimensional capillary-like networks observed at early time points suggest that PRP can also be used as an autologous cell delivery and pro-angiogenic system for bone tissue repair

Summary Statement. This experimental study showed that platelet rich fibrin matrix can improve muscle regeneration and long-term vascularization without local adverse effects. Introduction. Even though muscle injuries are very common, few scientific data on their effective treatment exist. Growth Factors (GFs) may have a role in accelerating muscle repair processes and a currently available strategy for their delivery into the lesion site is the use of autologous platelet-rich plasma (PRP). The present study is focused on the use of Platelet Rich Fibrin Matrix (PRFM), as a source of GFs. Materials and Methods. Bilateral muscular lesions were created on the longissimus dorsi muscle of Wistar rats. One side of the lesion was filled with a PRFM while the contralateral was left untreated (controls). Animals were sacrificed at 5, 10, 40 and 60 days from surgery. Histological, immunohistochemical and histomorphometric analyses were performed to evaluate muscle regeneration, neovascularization, fibrosis and inflammation. The presence of metaplasic zones, calcifications and heterotopic ossification were also assessed. Results. PRFM treated muscles exhibited an improved muscular regeneration, an increase in neovascularization, and a slight reduction of fibrosis compared with controls. No differences were detected for inflammation. Metaplasia, ossification and heterotopic calcification were not detected. Conclusions. This preliminary morphological experimental study shows that PRFM use can improve muscle regeneration and long-term vascularization. Since autologous blood products are safe, PRFM may be a useful and handily product in clinical treatment of muscle injuries

Background. Post-traumatic immunosuppression (PTI) after surgery increases vulnerability to nosocomial infections, sepsis, and death. Knee arthroplasty offers a sterile clinical model to characterise PTI and explore its underlying mechanisms. Methods. This prospective non-randomised cohort study of primary total knee arthroplasty was approved by the Local Ethics Committee. Exclusion criteria included revision-arthroplasty, pre-existing infections, blood-transfusions, malignancy, and auto-immune disease. 48 recruited patients fell into two groups, the first received unwashed anti-coagulated autologous salvaged blood transfusions after surgery (ASBT cohort, n=25). The second received no salvaged blood transfusions (NSBT cohort, n=18). Venous blood was sampled pre-operatively and within 3–7 days post-operatively. Salvaged blood was sampled at one and six hours post-operatively. Biomarkers of immune status included: interleukins (IL) or cytokines (x15), chemokines (x3), Damage-Associated-Molecular-Patterns (DAMPS) (x5), anti-microbial proteins (x3), CD24, and Sialic-acid-binding-Immunoglobulin-type-Lectin-10 (Siglec-10). Results were expressed as fold-change over pre-operative values. Only significant changes are described. Results. Certain biomarkers associated with sterile trauma were common to all 43 patients, including supra-normal: IL-6, IL-1-Receptor-Antagonist, IL-8, Heat-Shock-Protein-70 (HSP70), Calprotectin, CD24 and Siglec-10. But, whereas in NSBT patients post-operative pro-inflammatory biomarkers were sub-normal consistent with PTI, they were supra-normal in ASBT patients implying its reversal. These PTI-biomarkers included: IL-1β, IL-2, IL-17A, Interferon-gamma (IFN-γ), Tumour-Necrosis-Factor-alpha (TNF-α), and Annexin-A2. Reversal of PTI by salvaged blood was further endorsed in ASBT by sub-normal levels of the anti-inflammatory biomarkers: IL-4, IL-5, IL-10, and IL-13. Salvaged blood analyses revealed sustained supra-normal levels of DAMPs, CD24 and Siglec-10; and increasingly elevated levels of cytokines and chemokines during the six hour collection period. Interestingly, plasma CD24, Siglec-10, HSP70 and Calprotectin levels were significantly correlated, implying physical association within the circulation. Conclusions. Several anti-inflammatory processes triggered by traumatised tissue induce systemic PTI, thereby increasing vulnerability to infections. Reversal of PTI by re-infusion of anti-coagulated salvaged blood suggests a novel source of immuno-stimulants

Objectives

The purpose of this study was to compare the results and complications of tibial lengthening over an intramedullary nail with treatment using the traditional Ilizarov method.

Platelet-leucocyte gel (PLG), a new biotechnological blood product, has hitherto been used primarily to treat chronic ulcers and to promote soft-tissue and bone regeneration in a wide range of medical fields. In this study, the antimicrobial efficacy of PLG against Staphylococcus aureus (ATCC 25923) was investigated in a rabbit model of osteomyelitis. Autologous PLG was injected into the tibial canal after inoculation with Staph. aureus. The prophylactic efficacy of PLG was evaluated by microbiological, radiological and histological examination. Animal groups included a treatment group that received systemic cefazolin and a control group that received no treatment.

Treatment with PLG or cefazolin significantly reduced radiological and histological severity scores compared to the control group. This result was confirmed by a significant reduction in the infection rate and the number of viable bacteria. Although not comparable to cefazolin, PLG exhibited antimicrobial efficacy in vivo and therefore represents a novel strategy to prevent bone infection in humans.