Sylvie Miot

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial.

BACKGROUND Autologous native cartilage from the nasal septum, ear, or rib is the standard material for surgical reconstruction of the nasal alar lobule after two-layer excision of non-melanoma skin cancer. We assessed whether engineered autologous cartilage grafts allow safe and functional alar lobule restoration. METHODS In a first-in-human trial, we recruited five patients at the University Hospital Basel (Basel, Switzerland). To be eligible, patients had to be aged at least 18 years and have a two-layer defect (≥50% size of alar subunit) after excision of non-melanoma skin cancer on the alar lobule. Chondrocytes (isolated from a 6 mm cartilage biopsy sample from the nasal septum harvested under local anaesthesia during collection of tumour biopsy sample) were expanded, seeded, and cultured with autologous serum onto collagen type I and type III membranes in the course of 4 weeks. The resulting engineered cartilage grafts (25 mm × 25 mm × 2 mm) were shaped intra-operatively and implanted after tumour excision under paramedian forehead or nasolabial flaps, as in standard reconstruction with native cartilage. During flap refinement after 6 months, we took biopsy samples of repair tissues and histologically analysed them. The primary outcomes were safety and feasibility of the procedure, assessed 12 months after reconstruction. At least 1 year after implantation, when reconstruction is typically stabilised, we assessed patient satisfaction and functional outcomes (alar cutaneous sensibility, structural stability, and respiratory flow rate). FINDINGS Between Dec 13, 2010, and Feb 6, 2012, we enrolled two women and three men aged 76-88 years. All engineered grafts contained a mixed hyaline and fibrous cartilage matrix. 6 months after implantation, reconstructed tissues displayed fibromuscular fatty structures typical of the alar lobule. After 1 year, all patients were satisfied with the aesthetic and functional outcomes and no adverse events had been recorded. Cutaneous sensibility and structural stability of the reconstructed area were clinically satisfactory, with adequate respiratory function. INTERPRETATION Autologous nasal cartilage tissues can be engineered and clinically used for functional restoration of alar lobules. Engineered cartilage should now be assessed for other challenging facial reconstructions. FUNDING Foundation of the Department of Surgery, University Hospital Basel; and Krebsliga beider Basel.

Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: Towards annulus fibrosus tissue engineering

A novel design of silk-based scaffold is developed using a custom-made winding machine, with fiber alignment resembling the anatomical criss-cross lamellar fibrous orientation features of the annulus fibrosus of the intervertebral disc. Crosslinking of silk fibroin fibers with chondroitin sulphate (CS) was introduced to impart superior biological functionality. The scaffolds, with or without CS, instructed alignment of expanded human chondrocytes and of the deposited extracellular matrix while supporting their chondrogenic redifferentiation. The presence of CS crosslinking could not induce statistically significant changes in the measured collagen or glycosaminoglycan content, but resulted in an increased construct stiffness. By offering the combined effect of cell/matrix alignment and chondrogenic support, the silk fibroin scaffolds developed with precise fiber orientation in lamellar form represent a suitable substrate for tissue engineering of the annulus fibrosus part of the intervertebral disc.

Natural/synthetic porous scaffold designs and properties for fibro-cartilaginous tissue engineering

The goal of this study was to produce and characterize the scaffolds by combining the advantages of both natural and synthetic polymers for engineering fibro-cartilaginous tissues. Porous three-dimensional composite scaffolds were produced based on glycosaminoglycans and hyaluronic acid (HYAFF11) reinforced with polycaprolactone. The mechanical properties of scaffolds were evaluated as a function of time and compared with those of scaffolds seeded with human chondrocytes (constructs) and cultured in vitro up to 6 weeks. The composite scaffolds had a porosity of 68% with interconnected macropores with average pore sizes of 200 μm, an equilibrium swelling of 350%, and a predominant elastic behavior, typical of a macromolecular gel. The composite constructs maintained chondrocyte phenotype and degraded with the deposition of macromolecules synthesized by the cells. The scaffold presented mechanical properties and the ability to dissipate energy similar to the fibro-cartilaginous tissue.

The role of 3D structure and protein conformation on the innate and adaptive immune responses to silk-based biomaterials

We have investigated monocyte and T cell responsiveness to silk based biomaterials of different physico-chemical characteristics. Here we report that untransformed CD14+ human monocytes respond to overnight exposure to silk fibroin-based biomaterials in tridimensional form by IL-1β and IL-6, but not IL-10 gene expression and protein production. In contrast, fibroin based materials in bidimensional form are unable to stimulate monocyte responsiveness. The elicitation of these effects critically requires contact between biomaterials and responding cells, is not sustained and becomes undetectable in longer term cultures. We also observed that NF-κβ and p38 MAP kinase play key roles in monocyte activation by silk-based biomaterials. On the other hand, fibroin based materials, irrespective of their physico-chemical characteristics appeared to be unable to induce the activation of peripheral blood T cells from healthy donors, as evaluated by the expression of activation markers and IFN-γ gene.

Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial

BACKGROUND Articular cartilage injuries have poor repair capacity, leading to progressive joint damage, and cannot be restored predictably by either conventional treatments or advanced therapies based on implantation of articular chondrocytes. Compared with articular chondrocytes, chondrocytes derived from the nasal septum have superior and more reproducible capacity to generate hyaline-like cartilage tissues, with the plasticity to adapt to a joint environment. We aimed to assess whether engineered autologous nasal chondrocyte-based cartilage grafts allow safe and functional restoration of knee cartilage defects. METHODS In a first-in-human trial, ten patients with symptomatic, post-traumatic, full-thickness cartilage lesions (2-6 cm2) on the femoral condyle or trochlea were treated at University Hospital Basel in Switzerland. Chondrocytes isolated from a 6 mm nasal septum biopsy specimen were expanded and cultured onto collagen membranes to engineer cartilage grafts (30 × 40 × 2 mm). The engineered tissues were implanted into the femoral defects via mini-arthrotomy and assessed up to 24 months after surgery. Primary outcomes were feasibility and safety of the procedure. Secondary outcomes included self-assessed clinical scores and MRI-based estimation of morphological and compositional quality of the repair tissue. This study is registered with ClinicalTrials.gov, number NCT01605201. The study is ongoing, with an approved extension to 25 patients. FINDINGS For every patient, it was feasible to manufacture cartilaginous grafts with nasal chondrocytes embedded in an extracellular matrix rich in glycosaminoglycan and type II collagen. Engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of defect filling and development of repair tissue approaching the composition of native cartilage. INTERPRETATION Hyaline-like cartilage tissues, engineered from autologous nasal chondrocytes, can be used clinically for repair of articular cartilage defects in the knee. Future studies are warranted to assess efficacy in large controlled trials and to investigate an extension of indications to early degenerative states or to other joints. FUNDING Deutsche Arthrose-Hilfe.

Precultivation of Engineered Human Nasal Cartilage Enhances the Mechanical Properties Relevant for Use in Facial Reconstructive Surgery

Objective:To investigate if precultivation of human engineered nasal cartilage grafts of clinically relevant size would increase the suture retention strength at implantation and the tensile and bending stiffness 2 weeks after implantation. Summary Background Information:To be used for reconstruction of nasal cartilage defects, engineered grafts need to be reliably sutured at implantation and resist to bending/tension forces about 2 weeks after surgery, when fixation is typically removed. Methods:Nasal septum chondrocytes from 4 donors were expanded for 2 passages and statically loaded on 15 × 5 × 2-mm size nonwoven meshes of esterified hyaluronan (Hyaff-11). Constructs were implanted for 2 weeks in nude mice between muscle fascia and subcutaneous tissue either directly after cell seeding or after 2 or 4 weeks of preculture in chondrogenic medium. Engineered tissues and native nasal cartilage were assessed histologically, biochemically, and biomechanically. Results:Engineered constructs reproducibly developed with culture time into cartilaginous tissues with increasing content of glycosaminoglycans and collagen type II. Suture retention strength was significantly higher (3.6 ± 2.2-fold) in 2-week precultured constructs than in freshly seeded meshes. Following in vivo implantation, tissues further developed and maintained the original scaffold size and shape. The bending stiffness was significantly higher (1.8 ± 0.8-fold) if constructs were precultured for 2 weeks than if they were directly implanted, whereas tensile stiffness was close to native cartilage in all groups. Conclusion:In our experimental setup, preculture for 2 weeks was necessary to engineer nasal cartilage grafts with enhanced mechanical properties relevant for clinical use in facial reconstructive surgery.

Modulation of chondrocyte phenotype for tissue engineering by designing the biologic-polymer carrier interface.

Therapeutic strategies based on cell and tissue engineering can be advanced by developing material substrates that effectively interrogate the biological compartment, with or without the complimentary local release of growth factors. Poly(ether ester) segmented copolymers were engineered as model material systems to elucidate the interfacial molecular events that govern the function of adhered cells. Surface chemistry was modulated by varying poly(ethylene glycol) (PEG) length and mole fraction with poly(butylene terephthalate) (PBT), leading to differential competitive protein adsorption of fibronectin and vitronectin from serum and consequently to different cell attachment modes. Adhesion within the hydrogel-like milieu of longer surface PEG was mediated via binding to the CD44 transmembrane receptor, rather than the RGD-integrin mechanism, whereas greater substrate-bound fibronectin resulted in cell adhesion via integrins. These adhesion modalities differentially impacted morphological cell phenotype (spread or spheroid) and the subsequent expression of mRNA transcripts (collagen types II, I) characteristic of phenotypically differentiated or dedifferentiated chondrocytes, respectively. These results demonstrate that materials can be designed to directly elicit the membrane bound receptor apparatus desired for downstream cellular response, without requiring exogenous biological growth factors to enable differentiated potential.

Lumican inhibits collagen deposition in tissue engineered cartilage

Lumican is a glycoprotein that is found in the extracellular matrix of many connective tissues, including cartilage. It is a member of the small leucine-rich repeat proteoglycans family and along with two others, decorin and fibromodulin, has the capacity to bind to fibrillar collagens and limit their growth. Cartilage tissue engineering provides a potential method for the production of three-dimensional tissue for implantation into eroded joints. Many studies have demonstrated the growth of cartilage in vitro. However in all cases, biochemical analysis of the tissue revealed a significant deficit in the collagen content. We have now tested the hypothesis that the reduced collagen accumulation in engineered cartilage is a result of over-expression of decorin, fibromodulin or lumican. We have found that the lumican gene and protein are both over-expressed in engineered compared to natural cartilage whereas this is not the case for decorin or fibromodulin. Using a small hairpin lumican antisense sequence we were able to knockdown the lumican gene and protein expression in chondrocytes being used for tissue engineering. This resulted in increased accumulation of type II collagen (the major collagen of cartilage) whilst there was no significant alteration in the proteoglycan content. Furthermore, the antisense knockdown of lumican resulted in an increase in the average collagen fibril diameter measured by transmission electron microscopy. These results suggest that lumican plays a pivotal role in the development of tissue engineered cartilage and that regulation of this protein may be important for the production of high-quality implants.

Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First‐in‐Man Study

Stromal Vascular Fraction (SVF) cells freshly isolated from adipose tissue include osteogenic‐ and vascular‐progenitors, yet their relevance in bone fracture healing is currently unknown. Here, we investigated whether human SVF cells directly contribute to the repair of experimental fractures in nude rats, and explored the feasibility/safety of their clinical use for augmentation of upper arm fractures in elderly individuals. Human SVF cells were loaded onto ceramic granules within fibrin gel and implanted in critical nude rat femoral fractures after locking‐plate osteosynthesis, with cell‐free grafts as control. After 8 weeks, only SVF‐treated fractures did not fail mechanically and displayed formation of ossicles at the repair site, with vascular and bone structures formed by human cells. The same materials combined with autologous SVF cells were then used to treat low‐energy proximal humeral fractures in 8 patients (64‐84 years old) along with standard open reduction and internal fixation. Graft manufacturing and implantation were compatible with intraoperative settings and led to no adverse reactions, thereby verifying feasibility/safety. Biopsies of the repair tissue after up to 12 months, upon plate revision or removal, demonstrated formation of bone ossicles, structurally disconnected and morphologically distinct from osteoconducted bone, suggesting the osteogenic nature of implanted SVF cells. We demonstrate that SVF cells, without expansion or exogenous priming, can spontaneously form bone tissue and vessel structures within a fracture‐microenvironment. The gained clinical insights into the biological functionality of the grafts, combined with their facile, intra‐operative manufacturing modality, warrant further tests of effectiveness in larger, controlled trials. Stem Cells 2016;34:2956–2966