Due to the presence of megakaryocytes, platelets and clotting factors, bone marrow aspirate (BMA) tends to coagulate. For the first time, starting from our previous studies on mesenchymal vertebral stem cells, it has been hypothesized that coagulated BMA represents a safe and effective autologous biological scaffold for bone regeneration in spinal surgery. The present research involved advanced preclinical in vitro models and the execution of a pilot clinical study. Evaluation of cell morphology, growth kinetics, immunophenotyping, clonogenicity, trilineage-differentiation, growth-factors and HOX and TALE gene expression were analyzed on clotted- and un-clotted human V-BMA. In parallel, a pilot clinical study on ten patients with degenerative spine diseases submitted to instrumented posterior arthrodesis, is ongoing to assess the ability of clotted-V-BMA to improve spinal fusion at 6- and 12-months follow-up. Results demonstrated that clotted-V-BMA have significantly higher growth-factor expression and mesenchymal stem cell (MSCs) viability, homogeneity, clonogenicity, and ability to differentiate towards the osteogenic phenotype than un-clotted-V-BMA. Clotted-V-BMA also highlighted significant reduced expression of PBX1 and of MEIS3 genes negatively involved in osteoblast maturation and differentiation. From December 2020, eight patients have already been enrolled with first promising results that will be finally evaluated in the next two months. The application of V-BMA-clot as carrier of progenitors and cytokines and as natural scaffold with a structural texture represents a point-of-care orthobiologic product to improve spinal fusion. Clinical application seems to be efficacy, and we will confirm and strengthen these data with the final results of the pilot clinical study

The purpose of this work was to create an in vitro model of tissue-engineered cartilage structure produced by isolated swine articular chondrocytes, expanded in culture and seeded onto a biological scaffold. Swine articular chondrocytes were enzymatically isolated from pig joints and expanded in monolayer culture. When confluence was reached, cells were resuspended and seeded in vitro onto biological collagen scaffolds for 3, 4 and 6 weeks. Samples were retrieved from the culture and analysed macroscopically and biomechanically by compressive test. Gross evaluation was performed by simple probing, sizing and weighing the samples at all time periods. A baseline of the values was also recorded at time 0. Then, samples were biomechanically tested by unconfined compression and shear tests. Finally, the samples were fixed in 4% paraformaldehyde and processed for histological evaluation. Some samples were stained with Safranin-o, and some others subjected to immunostaining analysis for type II collagen. Upon retrieval, samples showed dimensional enlargement and mass increase over time and gross mechanic integrity by simple probing. A biomechanical test demonstrated an initial reduction in the values of compressive and shear parameters, followed by a consistent increase throughout the tested time points. Histology showed cartilage-like tissue maturing over time within the biological scaffold. The results from this study demonstrate that isolated chondrocytes could be seeded onto a biological collagen scaffold, producing cartilage-like matrix with tissue-specific morphology and biomechanical integrity. This tissue-engineered cartilage structure is easily reproducible and it could represent a valuable model for studying the behaviour of different variables on the newly formed cartilage

Human mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate into mesoderm-type cells such as osteoblasts, chondroblast, tenocytes etc. They can be retrieved by different sources, but the number of cells obtained suggested the adipose tissue as a primary harvest site of MSCs. Cells can be harvested using the Coleman procedure, obtaining stromal vascular fraction (SVF), enriched with MSCs, after collagenase digestion. The availability of SVF storage has been envisioned for multiple treatments of the degenerated tissue. Indeed, the use of SVF has been introduced into clinical trials for tissue regeneration for orthopaedic patients. Difficulties of a selective delivery of SVF locally have been previously discussed. Thus, the use of biological scaffolds in order to better localize SVF in the tissue site has been studied. The methodological evolution for the use of SVF in the best possible biological conditions is a milestone for good clinical results

The growth in the popularity of tissue engineering principles in the treatment of musculoskeletal disorders has been complemented greatly with research investment into tissue specific scaffolds. Biological scaffolds produced by means of decellularising native tissues have the advantage of providing the natural complex hierarchical matrix and, in doing so, replicating the specific biomechanical and biological functions of the tissue in question. Decellularisation treatments are multi-faceted, vary considerably between different processes and may involve many lengthy treatment steps. Some of these bio-processes may cause undesirable structural changes to the extracellular matrix of tissues and, by association, their mechanical properties. Thus, it is of paramount importance to ensure that the properties of the scaffolds are not affected to the extent of reducing their integration, biomechanical performance and longevity. This talk consists of a body of work detailing investigations into bio-process optimisation, sterilisation strategies and the regenerative and functional capacity of decellularised xenogeneic and allogeneic tendon, ligament and bone scaffolds. In addition, on-going work concerning advanced pre-clinical assessment, stratification of these products to particular patient populations and the importance of the manufacturing value chain in their translation will be discussed

Aims

Orthopaedic surgery requires grafts with sufficient mechanical strength. For this purpose, decellularized tissue is an available option that lacks the complications of autologous tissue. However, it is not widely used in orthopaedic surgeries. This study investigated clinical trials of the use of decellularized tissue grafts in orthopaedic surgery.

Bioactive glasses, such as 45S5 Bioglass (BG), have been shown to promote bone ingrowth both in vitro and in vivo. The goal of this study was to analyze the effect of a high dose of BG (20%) in Direct Ink Writing (DIW)-produced controlled-geometry PCL-BG composite scaffolds in both their mechanical and biological performance. Porous cubes of 5 × 5 × 5 mm, 50% porosity and pore size and strut diameter of 400 µm were fabricated in a 3D-Bioplotter (EnvisionTec) to investigate their biological performance (n = 3). Additionally, cylindrical specimens (10 mm diameter; 15 mm height) with same internal structure were fabricated for mechanical testing (n = 6). The cylindrical specimens were tested by compression in a universal testing machine (ZwickRoell) with a 10 kN load cell. The tests were performed at 1.00 mm/min with extensometers in both sides. For biological characterization, scaffolds were sterilized in 70% ethanol overnight and pre-incubated with DMEM for 1 hour at room temperature. 1×10. 5. human gingival mesenchymal stem cells (hGMSCs) in 50 µl DMEM were seeded on the scaffolds using agarose molds to improve cell adhesion, and cultured in standard cell-culture conditions for 3, 7 and 14 days. To measure cell proliferation, the reagent CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega) was added to the cell-seeded scaffolds at each time point, using non-seeded scaffolds as blank controls. The OD (490 nm) was measured in a BioTek 800 TS plate reader. Both the apparent elastic modulus and yield stress were significantly lower in the scaffolds with 20% BG than their PCL control counterparts (p < 0.0001 for elastic modulus and p < 0.005 for yield stress, t-test). Cell proliferation in the scaffolds by MTS was variable, with the 20% BG scaffolds showing a significantly higher signal after seven days in culture (p < 0.05 by t-test), but a significantly lower signal after 14 days in culture (p < 0.05 by t-test). In conclusion, scaffolds with 20% BG showed a lower mechanical performance than their PCL counterparts in terms of both their apparent elastic modulus and yield stress. Additionally, scaffolds with 20% BG showed variable cell proliferation rates in terms of their metabolic activity over a two-week period. The decrease in proliferation rate after week 2 after an initial increase at the end of week 1 could be due to cytotoxic effects of the BG at this high dose (20%) after long term exposure. These results suggest that a dosage of 20% BG may not necessarily improve the mechanical and biological performance of scaffolds, so future experiments are required in order to characterize the optimum BG dosage in PCL scaffolds for tissue engineering applications

Introduction. Tendon injuries remain challenging, secondary healing and prolonged immobilisation result in suboptimal outcome. Previous study by our group showed that demineralised bone matrix (DBM) can result in faster healing of a tendon enthesis. The aim of this study is to test different ways augmenting tendon with DBM to enhance tendon repair and regeneration. Methods. DBM strips were prepared from tibias of mature ewes. Patella, patellar tendon and tibias were dissected and the distal 1 cm of the patellar tendon was excised. 4 models were designed;. Model-1, DBM strip was used to bridge the gap between the tendon and the tibial tuberosity. The DBM strip was stitched to the tendon using one bone anchor. Model-2, similar to model 1 with the use of 2 anchors. Model-3, similar to model 2, construct was off loaded by continuous thread looped twice through bony tunnels sited in the patella and in the tibial tuberosity. Model-4, similar to model 3 with 3 threads as off loading loop. All models were tested for pullout force and mode of failure. Results. The median failure force for model-1 (N=5) was 250N while for model-2 (N=5) was 290N. In model-3 and model-4 failure of the off loading loop was used as end point, 6 samples were tested in each model. Median failure force of model-3 was 767N and for model-4 was 934N. There was no statistical significance between model-1 and model-2 (p=0.249), however statistical significance was found between other models (p=< 0.006). Discussion. A study published in 1996 proved that cortical DBM can be used as ACL graft with evidence of ligamentisation. DBM provides a biologic scaffold with potential for use as ligament and tendon replacement. Our study shows that a tendon rupture can be augmented with DBM giving intial appropriate mechanical strength suitable for in-vivo use

Introduction: Current treatment options for small, contained articular cartilage defects include microfracture, osteochondral autograft plugs or newer synthetic plugs. Chondromimetic is a novel biphasic biological scaffold composed of collagen and glycosaminoglycan. The addition of brushite provides the scaffold with a regionally specific component mimicking both phases of the osteochondral unit. The aim of this study was to show the efficacy of Chondromimetic in repairing a surgically created osteochondral defect in a caprine model. Methods: Osteochondral defects were made in the lateral trochlear sulcus (LTS) and medial femoral condyle (MFC) of nine goats. Chondromimetic scaffolds (6x6mm) were inserted into each defect (n=6), while three controls had defects left empty (n=3). All animals were sacrificed at 26 weeks postoperatively. Macroscopic evaluations and quantitative stiffness properties were assessed. Histological sections were taken at approximately the centre of the defect, stained with Safrinin O/Fast Green and scored using a validated quantitative assessment tool. Results: Macroscopically, the repair tissue scored higher in the MFC and LTS (p< 0.05) compared to controls. In all defects, the mechanical stiffness was found to be within one standard deviation of native cartilage, except that of the LTS controls. Histologically, the predominant tissue in the cartilage layer was deemed to be hyaline-like in three of six MFC defects, and five of six LTS defects according to the modified Sellers score. This was compared to one in three and zero of three in the MFC and LTS controls respectively. Discussion: These results represent the early findings from an ongoing in-vivo study in which a further group of animals will be sacrificed at one year. At six months, the histology and mechanical properties are encouraging and should continue to improve with time. These results show that Chondromimetic may represent an acceptable alternative to marrow stimulation in the treatment of osteochondral defects

Introduction: Current treatment options for small, contained articular cartilage defects include microfracture, osteochondral autograft plugs or newer synthetic plugs. Chondromimetic is a novel biphasic biological scaffold composed of collagen and glycosaminoglycan. The addition of brushite provides the scaffold with a regionally specific component enabling the scaffold to mimic both phases of the osteochondral unit. The aim of this study was to show the efficacy of Chondromimetic in repairing a surgically created osteochondral defect in a caprine model. Methods: Osteochondral defects were made in the lateral trochlear sulcus (LTS) and medial femoral condyle (MFC) of nine goats. Chondromimetic scaffolds (6x6mm) were inserted into each defect (n=6), while three controls had defects left empty (n=3). All animals were sacrificed at 26 weeks postoperatively. Macroscopic evaluations and quantitative stiffness properties were assessed. Histological sections were taken at approximately the centre of the defect, stained with Safrinin O/Fast Green and scored using a validated quantitative assessment tool. Results: Macroscopically, the repair tissue scored higher in the filled MFC and LTS (p< 0.05) compared to controls. In all defects, the mechanical stiffness was found to be within one standard deviation of native cartilage, except the LTS controls. Histologically, the predominant tissue in the cartilage layer was hyaline-like in three of six filled MFC defects, and five of six filled LTS defects according to the modified Sellers score. This was compared to one in three and zero of three in the MFC and LTS controls respectively. Discussion: These results represent the early findings from an ongoing in-vivo study in which a further group of animals will be sacrificed at one year. At six months, the histology and mechanical properties are encouraging and should continue to improve with time. These results show that Chondromimetic may represent an acceptable alternative to marrow stimulation in the treatment of osteochondral defects

Introduction: Homogenous cell distribution and suffi-cient initial scaffold stability remain key issues for successful tissue engineered osteochondral constructs. The purpose of this study was to investigate the application of initial compression forces during the first 24 hours of cell culture followed by different stress patterns. Methods: Bone marrow stromal cells were harvested from the iliac crest during routine trauma surgery. The cells were expanded in a 2-dimensional culture and then seeded into the biologic hybrid scaffold with a concentration of 1x10E6 cells per ml. Pressure and vacuum forces were applied in a specially developed glass kit. The constructs were exposed to two different protocols of compression combined as oteochondral matrices of CaReS (collagen I) and Tutobone (Ars Arthro, Esslingen, Germany and Tutogen Medical GmbH, Neunkirchen a. Br., Germany). Controls were resected osteochondral fragments from patients with articular fractures and uncompressed constructs. These effects were evaluated using light microscopy after standard staining to identify matrix penetration. Biomechanical tests were conducted, too using a modified biomechanical testing machine. The ‘constrained compression’, maximum load to failure, modulus, and strain energy density were determined. Results: Histology: Penetration and cell distribution was demonstrated homogenous and vital, respectively. Mechanical tests showed a significant enhancement of primary matrix stability. The following stress patterns did not enhance significantly stability over seven days. Discussion: The aim of this project was to investigate the response and cell distrubution of human bone marrow stromal cells seeded on a 3-dimensional biologic hybrid scaffold using compression and vacuum forces. The integration of mechanical stimulation in the tissue engineering process may lead to a progress in the structural and biomechanical properties of these tissues and offers new possibilities in the management of bone injuries and degenerative diseases

Aims

The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects.

Introduction. Avulsion of abductors from hip is a debilitating complication after total hip arthroplasty performed through a trans-gluteal approach. It results in intractable pain, Trendelenberg limp and instability of the hip. Techniques described for repairing these abductor tears including direct trans osseous repairs, endoscopic repair techniques, Achilles tendon allograft, Gluteus Maximus and Vastus Lateralis muscle transfers. The aim of our study was to assess improvement in pain, limp and abductor strength in patients operated upon surgically for confirmed abductor avulsion using a modified trans osseous repair and augmentation of repair with a Graft Jacket allograft acellular human dermal matrix (Graft jacket; Wright Medical Technology, Arlington, TN). Patients and Methods. In this prospective study we include 18 consecutive patients with hip abductor avulsions following a primary total hip arthroplasty through Hardinge approach for osteoarthritis. All the patients presented with pain around lateral aspect of hip, walking with a significant Trendelenberg limp and used a crutch or a stick in the opposite hand. Diagnosis was made by clinical examination and confirmed by MRI scans. Surgical Technique. Surgical procedure was through lateral approach using the old scar to mobilise combined aponeurosis of the Gluteal Medius and Gluteus Minimus from the bony bed on the ilium to permit advancement onto the trochanter. The conjoint Gluteus Medius and Minimus insertion was affixed to the greater trochanter with No.5 non absorbable trans-osseous suture using a Krackow stitch through a series of transverse tunnels made in anterior aspect of greater trochanter. An on lay augmentation (Fig. 1) of the osseo-tendinous junction was performed using a Graft Jacket matrix of 4 × 7 cms in size after rehydration according to the manufacturer's instructions. Results. At mean follow up of 22 months (15–34 months), pain improved in all patients with mean VAS score improving from 8.25 to 2.33 (p value-0.05). All the patients had improvement in their abductor strength with MRC grade 4 out of 5 in 16 patients and 3 out of 5 in 2 patients. Trendelenberg sign disappeared in all but two. Mean Harris hip score improved from 34.05 to 81.26 (p value-0.001). All patients had improvement in gait except mild noticeable limp in two patients.13 patients did not use any walking aids and five felt more secure using a walking stick in the contralateral hand. The mean SF-36 Physical component score was 53.47 and Mental component score was 56.07. Conclusions. The procedure is safe and associated with high patient satisfaction, without the morbidity of tendon or muscle transfers. The Graft Jacket Matrix provides biological bridging between the hip abductors and its insertion into greater trochanter. It provides a biological scaffold for cellular and vascular in-growth and constructive tissue remodelling. The described procedure appears to enhance the mechanical strength of repaired tendon immediately following surgery. The Graft Jacket allograft matrix has already been used successfully in rotator cuff repairs of shoulder and has showed encouraging results. The early success of this new procedure warrants further study with more patients, longer follow-up and possibly histological study of retrieved specimens

Introduction: The aim of this study was to develop a technique to decellularise a porcine cartilagebone construct with a view to using this as a biological scaffold for transplantation into human osteochondral defect as a cartilage substitute. Methods: Decellularisation was based on a modification of the technique of Booth et al (2002). Cartilage bone matrix (n=9) were decellularised by exposing the tissue to 2 cycles of dry freeze-thaw followed 2 more cycles with the addition of hypotonic (10mM tris-HCl, pH8.0) buffer. Samples were then cycled through hypotonic buffer, followed by ionic detergent (0.1% [w/v] sodium dodecyl sulphate [SDS]) in the presence of protease inhibitors (aprotinin 10 KIU/ml) and 0.1% (w/v) ethylene diamine tetraacetic acid (EDTA). This was followed by washes in PBS with aprotinin and incubation in nuclease solution containing DNase (50U/ml) and RNase (1U/ml). Decontamination using 0.1% (v/v) peracetic acid in PBS was then incorporated to achieve disinfection of the tissue samples. Finally, samples were washed in PBS. Three decellularisation protocols were used depending on the number of hypotonic/SDS cycles: this was either done once, three or six times referred to as DC1, DC3 and DC6 respectively. Fresh & decellularised cartilage were compared histologically using haematoxylin and eosin staining, to visualize cellular content, sirius red, to visualise collagen fibres & alcian blue, to visualise glycosaminoglycans (GAG). Immunohistochemistry staining for galactose-α-1,3-galactose (α-gal), collagen I, II & VI was performed for fresh and decellularised samples. DNA assay: Genomic DNA was extracted using a DNA isolation kit for tissues (Roche Applied Sciences). Collagen and DMB sulphated sugar assay, as described by Stapleton et al. (2008), were performed to measure collagen and GAG content. The biphasic property of fresh and decellularised cartilage was determined using a pin on plate indentation test. Results: H& E staining revealed the absence of visible whole cells. Sirius red stain gave evidence of the retention of collagen following decellularisation. In contrast, the acellular matrix showed evidence of loss of GAGs. There was no evidence of the expression of α-gal in the acellular scaffold. DNA analysis revealed the absence of genomic DNA in comparison to fresh tissues (ANOVA, p< 0.05). The decellularisation process had minimal effect on the collagen content of the cartilage. Nevertheless there was a significant difference in the sulphated sugar content of the fresh tissue when compared to the decellularised tissue (ANOVA, p< 0.05), indicating loss of 92% GAG. Biomechanical testing of decellularised tissues showed a significant change (ANOVA, p< 0.05) in comparison to the fresh cartilage. Discussion: In conclusion this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair. To our knowledge, this is the first study that has successfully removed whole cells and α-gal from xenogeneic cartilage and bone tissue. Future studies are required to investigate methods to recellularise the acellular matrix using an appropriate cell type and mechanical conditioning and to investigate replenishing GAG loss following decellularisation

Objectives

This study investigated the biomechanical performance of decellularized porcine superflexor tendon (pSFT) grafts of varying diameters when utilized in conjunction with contemporary ACL graft fixation systems. This aimed to produce a range of ‘off-the-shelf’ products with predictable mechanical performance, depending on the individual requirements of the patient.

Aims

To evaluate graft healing of decellularized porcine superflexor tendon (pSFT) xenograft in an ovine anterior cruciate ligament (ACL) reconstruction model using two femoral fixation devices. Also, to determine if pSFT allows functional recovery of gait as compared with the preoperative measurements.

Introduction: Bone is unique with a vast potential for regeneration from cells with stem cell characteristics. With an increasing aging population, clinical imperatives to augment and facilitate tissue repair have highlighted the therapeutic potential of harnessing mes-enchymal populations from bone. We describe laboratory and clinical findings from two clinical cases, where different proximal femoral conditions (AVN, bone cyst) were treated with impacted allograft augmented with marrow-derived allogeneic progenitor cells. Methods: Marrow was aspirated from the posterior superior iliac crest and seeded onto prepared washed morsellised allograft. The seeded graft was left for 40 minutes to allow adherence of the marrow-derived osteoprogenitor cells prior to impaction into the defect. Samples of the impacted graft were taken for in-vitro analysis of cell viability, histology and biochemical analysis of cell number and osteogenic enzyme activity. The total force imparted during impaction was calculated using a load cell, with three independent surgeons performing a laboratory simulation of the impaction technique. Results: Both patients made a rapid clinical recovery after an overnight stay. Imaging confirmed filling of the defects with increased density on plain radiographs suggesting good impaction of the graft composite. Immu-nohistochemical staining of graft samples demonstrated that a living composite graft with osteogenic activity had been introduced into the defects as evidenced by cell tracker green viability and alkaline phosphatase (osteogenic marker) expression and specific activity. The average peak forces during impaction were 0.7kN corresponding to average peak stresses within the graft of 8.3MPa. Similar forces were seen between operators. Discussion: Replacement of bone loss remains a major challenge in orthopaedic practice. Although allograft remains the gold standard where large volumes preclude autograft, allograft has little osteoinductive potential. We demonstrate that marrow-derived cells can adhere to highly washed morsellised allograft, survive the impaction process, and are of the osteoblastic phenotype creating a living composite. Thus we conclude, impacted allograft seeded with autologous marrow cells allows the delivery of a biologically active scaffold for the treatment of bone deficiency. In addition this is a novel straightforward technique, surgeon independent and with applications in a number of orthopaedic scenarios

Objectives

Recently, the field of tissue engineering has made numerous advances towards achieving artificial tendon substitutes with excellent mechanical and histological properties, and has had some promising experimental results. The purpose of this systematic review is to assess the efficacy of tissue engineering in the treatment of tendon injuries.

Objectives

The objective of this study was to determine if the use of fascia lata as a tendon regeneration guide (placed into the tendon canal following harvesting the semitendinosus tendon) would improve the incidence of tissue regeneration and prevent fatty degeneration of the semitendinosus muscle.