Osnat Hakimi

Synthetic and degradable patches: an emerging solution for rotator cuff repair.

The use of rotator cuff augmentation has increased dramatically over the last 10 years in response to the high rate of failure observed after non‐augmented surgery. However, although augmentations have been shown to reduce shoulder pain, there is no consensus or clear guideline as to what is the safest or most efficacious material. Current augmentations, either available commercially or in development, can be classified into three categories: non‐degradable structures, extra cellular matrix (ECM)‐based patches and degradable synthetic scaffolds. Non‐degradable structures have excellent mechanical properties, but can cause problems of infection and loss of integrity in the long‐term. ECM‐based patches usually demonstrate excellent biological properties in vitro, but studies have highlighted complications in vivo due to poor mechanical support and to infection or inflammation. Degradable synthetic scaffolds represent the new generation of implants. It is proposed that a combination of good mechanical properties, active promotion of biological healing, low infection risk and bio‐absorption are the ideal characteristics of an augmentation material. Among the materials with these features, those processed by electrospinning have shown great promis. However, their clinical effectiveness has yet to be proven and well conducted clinical trials are urgently required.

A layered electrospun and woven surgical scaffold to enhance endogenous tendon repair.

Surgical reattachments of tendon to bone in the rotator cuff are reported to fail in around 40% of cases. There are no adequate solutions to improve tendon healing currently available. Electrospun, sub-micron materials, have been extensively studied as scaffolds for tendon repair with promising results, but are too weak to be surgically implanted or to mechanically support the healing tendon. To address this, we developed a bonding technique that enables the processing of electrospun sheets into multi-layered, robust, implantable fabrics. Here, we show a first prototype scaffold created with this method, where an electrospun sheet was reinforced with a woven layer. The resulting scaffold presents a maximum suture pull out strength of 167N, closely matched with human rotator cuff tendons, and the desired nanofibre-mediated bioactivity in vitro and in vivo. This type of scaffold has potential for broader application for augmenting other soft tissues.

Fabrication of continuous electrospun filaments with potential for use as medical fibres.

Soft tissue injuries represent a substantial and growing social and economic burden. Medical fibres are commonly used to repair these injuries during surgery. Patients outcomes are, however, not promising with around 40% of surgical repairs failing within the first few months after surgery due to poor tissue regeneration. The application of nanofibrous filaments and yarns as medical fibres and scaffolds has been suggested to improve soft tissue regeneration and enhance the quality of the repair. However, due to a lack of robustness and reliability of the current fabrication methods, continuous nanofibrous filaments cannot be manufactured and scaled up in industrial settings and are not currently available for clinical use. We have developed a robust and automated method that enables the manufacture of continuous electrospun filaments and which has the potential to be integrated into existing textile production lines. The technology uses a wire guide to form submicrofibres in a dense, narrow mesh which can be detached as a long and continuous thread. The thread can then be stretched and used to create multifilament yarns which can imitate the hierarchical architecture of tissues such as tendons and ligaments. Electrospun polydioxanone yarns produced by this method showed improved cellular proliferation and adhesion when compared to medical monofilament fibres in current clinical use. In vivo, the electrospun yarns showed a good safety profile with mild foreign body reaction and complete degradation within 5 months after implantation. These results suggest that this filament collection method has the potential to become a useful platform for the fabrication of future medical textiles.

Distribution and expression of type VI collagen and elastic fibers in human rotator cuff tendon tears.

Abstract There is increasing evidence for a progressive extracellular matrix change in rotator cuff disease progression. Directly surrounding the cell is the pericellular matrix, where assembly of matrix aggregates typically occurs making it critical in the response of tendon cells to pathological conditions. Studies in animal models have identified type VI collagen, fibrillin-1 and elastin to be located in the pericellular matrix of tendon and contribute in maintaining the structural and biomechanical integrity of tendon. However, there have been no reports on the localization of these proteins in human tendon biopsies. This study aimed to characterize the distribution of these ECM components in human rotator cuffs and gain greater insight into the relationship of pathology to tear size by analyzing the distribution and expression profiles of these ECM components. Confocal microscopy confirmed the localization of these structural molecules in the pericellular matrix of the human rotator cuff. Tendon degeneration led to an increased visibility of these components with a significant disorganization in the distribution of type VI collagen. At the genetic level, an increase in tear size was linked to an increased transcription of type VI collagen and fibrillin-1 with no significant alteration in the elastin levels. This is the first study to confirm the localization of type VI collagen, elastin and fibrillin-1 in the pericellular region of human supraspinatus tendon and assesses the effect of tendon degeneration on these structures, thus providing a useful insight into the composition of human rotator cuff tears which can be instrumental in predicting disease prognosis.

Gene expression profiles of changes underlying different-sized human rotator cuff tendon tears

BACKGROUND Progressive cellular and extracellular matrix (ECM) changes related to age and disease severity have been demonstrated in rotator cuff tendon tears. Larger rotator cuff tears demonstrate structural abnormalities that potentially adversely influence healing potential. This study aimed to gain greater insight into the relationship of pathologic changes to tear size by analyzing gene expression profiles from normal rotator cuff tendons, small rotator cuff tears, and large rotator cuff tears. METHODS We analyzed gene expression profiles of 28 human rotator cuff tendons using microarrays representing the entire genome; 11 large and 5 small torn rotator cuff tendon specimens were obtained intraoperatively from tear edges, which we compared with 12 age-matched normal controls. We performed real-time polymerase chain reaction and immunohistochemistry for validation. RESULTS Torn rotator cuff tendons demonstrated upregulation of a number of key genes, such as matrix metalloproteinase 3, 10, 12, 13, 15, 21, and 25; a disintegrin and metalloproteinase (ADAM) 12, 15, and 22; and aggrecan. Amyloid was downregulated in all tears. Small tears displayed upregulation of bone morphogenetic protein 5. Chemokines and cytokines that may play a role in chemotaxis were altered; interleukins 3, 10, 13, and 15 were upregulated in tears, whereas interleukins 1, 8, 11, 18, and 27 were downregulated. CONCLUSIONS The gene expression profiles of normal controls and small and large rotator cuff tear groups differ significantly. Extracellular matrix remodeling genes were found to contribute to rotator cuff tear pathogenesis. Rotator cuff tears displayed upregulation of a number of matrix metalloproteinase (3, 10, 12, 13, 15, 21, and 25), a disintegrin and metalloproteinase (ADAM 12, 15, and 22) genes, and downregulation of some interleukins (1, 8, and 27), which play important roles in chemotaxis. These gene products may potentially have a role as biomarkers of failure of healing or therapeutic targets to improve tendon healing.

Repairing damaged tendon and muscle: are mesenchymal stem cells and scaffolds the answer?

Mesenchymal stem cells (MSCs) have become an area of intense interest in the treatment of musculoskeletal conditions, such as muscle and tendon injury, as various animal and human trials have demonstrated that implantation with MSCs leads to improved healing and function. However, these trials have usually been relatively small scale and lacking in adequate controls. Additionally, the optimum source of these cells has yet to be determined, partly due to a lack of understanding as to how MSCs produce their beneficial effects when implanted. Scaffolds have been shown to improve tissue-engineering repairs but require further work to optimize their interactions with both native tissue and implanted MSCs. Robust, well-controlled trials are therefore required to determine the usefulness of MSCs in musculoskeletal tissue repair.

Identifying the optimum source of mesenchymal stem cells for use in knee surgery.

Single sitting procedures where the mononuclear cell fraction is extracted from bone marrow and implanted directly into cartilage and bone defects are becoming more popular as novel treatments for cartilage defects which have, until now had few treatment options. This is on the basis that the mesenchymal stem cells (MSCs) contained within will repair the damaged tissue. This study sought to determine if the femur and tibia could provide equivalent amounts of mesenchymal stem cells, with equivalent viability and proliferative capacity, to that obtained from the gold standard of the pelvis in order to potentially reduce the morbidity associated with these procedures. Bone marrow was extracted from the pelvis, femur, and tibia of human subjects. The mononuclear cell fraction was extracted and cultured in the laboratory. Mesenchymal stem cell populations were assessed using a colony forming unit count. Viability was assessed using a PrestoBlue viability assay. Population doubling number was calculated between the end of passage 0 and passage three to determine the proliferative abilities of the different populations. Finally, the cell surface phenotype of the cells was determined by flow cytometry. The results showed that the pelvis was superior to the femur and tibia in terms of the number of stem cells isolated. There was no statistically significant difference in the phenotype of the cells isolated from different locations. This work shows that when undertaking single sitting procedures, the pelvis remains the optimum source for obtaining MSCs, despite the morbidity associated with bone marrow collection from the pelvis.

Differential growth on sutures of tendon cells derived from torn human rotator cuff

Rotator cuff tendon pathology is proposed to account for 30-70% of all shoulder pain and surgical repair with a nonabsorbable suture is the common option for painful rotator cuff tears that have failed conservative treatment. A number of studies have suggested the beneficial effect of augmenting the repair with implants constructed from polymers used for sutures. Thus, it was of interest to investigate the affinity of tendon-derived fibroblasts, often thought to be the repairing agents of torn tendons, to commonly used sutures. The aim of this comparative study was to evaluate the suitability of these sutures for the construction of a patch by measuring cell survival, proliferation, and migration of human tendon-derived fibroblasts on different sutures. To ensure relevance to the target tissue, cells used in this study were obtained from torn human supraspinatus tendons. An initial comparison of cell proliferation on suture mats showed an overall positive proliferation on polyester (Ethibond) and polydioxanone (PDSII) mats and a reduction of proliferation on vicryl (polyglactin 910) compared to day one. The results also showed that the degradation products of vicryl had a negative effect on cell growth over 10 weeks. Of the commercial sutures selected and tested, Ethibond showed the best performance in terms of cell attachment and increase in biomass. The degradable PDSII also showed good interaction with cells in vitro, but relatively poor cell adhesion. This study provides useful and clinically relevant information, which could help to guide future considerations for candidate materials from which to construct tissue repair patches.

A quantitative label-free analysis of the extracellular proteome of human supraspinatus tendon reveals damage to the pericellular and elastic fibre niches in torn and aged tissue.

Tears of the human supraspinatus tendon are common and often cause painful and debilitating loss of function. Progressive failure of the tendon leading to structural abnormality and tearing is accompanied by numerous cellular and extra-cellular matrix (ECM) changes in the tendon tissue. This proteomics study aimed to compare torn and aged rotator cuff tissue to young and healthy tissue, and provide the first ECM inventory of human supraspinatus tendon generated using label-free quantitative LC-MS/MS. Employing two digestion protocols (trypsin and elastase), we analysed grain-sized tendon supraspinatus biopsies from older patients with torn tendons and from healthy, young controls. Our findings confirm measurable degradation of collagen fibrils and associated proteins in old and torn tendons, suggesting a significant loss of tissue organisation. A particularly marked reduction of cartilage oligomeric matrix protein (COMP) raises the possibility of using changes in levels of this glycoprotein as a marker of abnormal tissue, as previously suggested in horse models. Surprisingly, and despite using an elastase digestion for validation, elastin was not detected, suggesting that it is not highly abundant in human supraspinatus tendon as previously thought. Finally, we identified marked changes to the elastic fibre, fibrillin-rich niche and the pericellular matrix. Further investigation of these regions may yield other potential biomarkers and help to explain detrimental cellular processes associated with tendon ageing and tendinopathy.

A comparative evaluation of the effect of polymer chemistry and fiber orientation on mesenchymal stem cell differentiation.

Abstract Bioengineered tissue scaffolds in combination with cells hold great promise for tissue regeneration. The aim of this study was to determine how the chemistry and fiber orientation of engineered scaffolds affect the differentiation of mesenchymal stem cells (MSCs). Adipogenic, chondrogenic, and osteogenic differentiation on aligned and randomly orientated electrospun scaffolds of Poly (lactic‐co‐glycolic) acid (PLGA) and Polydioxanone (PDO) were compared. MSCs were seeded onto scaffolds and cultured for 14 days under adipogenic‐, chondrogenic‐, or osteogenic‐inducing conditions. Cell viability was assessed by alamarBlue metabolic activity assays and gene expression was determined by qRT‐PCR. Cell‐scaffold interactions were visualized using fluorescence and scanning electron microscopy. Cells grew in response to scaffold fiber orientation and cell viability, cell coverage, and gene expression analysis showed that PDO supports greater multilineage differentiation of MSCs. An aligned PDO scaffold supports highest adipogenic and osteogenic differentiation whereas fiber orientation did not have a consistent effect on chondrogenesis. Electrospun scaffolds, selected on the basis of fiber chemistry and alignment parameters could provide great therapeutic potential for restoration of fat, cartilage, and bone tissue. This study supports the continued investigation of an electrospun PDO scaffold for tissue repair and regeneration and highlights the potential of optimizing fiber orientation for improved utility.