Marcel Jakob

Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF‐2, TGFβ1, or FGF‐2/TGFβ1). Cells were then induced to redifferentiate in 3D pellets using serum‐supplemented medium (SSM), serum‐free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGFβ1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real‐time PCR. FGF‐2/TGFβ1‐expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR‐expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF‐, PDGFbb‐ or FGF‐2‐expanded chondrocytes. Upon supplementation of SFM with TGFβ and dexamethasone (SFM TD), CII/CI ratios decreased 4.4‐fold for EGF‐ and PDGFbb‐expanded chondrocytes, but increased 96‐fold for FGF‐2/TGFβ1‐expanded cells. Chondrocytes expanded with FGF‐2/TGFβ1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment. J. Cell. Biochem. 81:368–377, 2001.

Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real‐time RT‐PCR assays to investigate how the expression of typical osteoblast‐related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor‐2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, β‐glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein‐2 (BMP‐2), bone sialo protein‐II (BSP), osteopontin (OP) and to a lower extent cbfa‐1 increased with culture time in osteogenic medium (OM), both in CTR‐ and FGF/Dex‐expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP‐2, BSP, and OP were more expressed in FGF/Dex than in CTR‐expanded BMSC (mRNA levels were, respectively, 9.5‐, 14.9‐, and 5.8‐fold higher), unlike all the other investigated genes. Analysis of single‐colony‐derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex‐expanded clones expressed higher mRNA levels of BMP‐2, BSP, and OP than CTR‐expanded clones. In conclusion, we provide evidence that mRNA levels of BMP‐2, BSP, and OP, quantified using real‐time RT‐PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion. J. Cell. Biochem. 85: 737–746, 2002.

Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts

Three‐dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor‐based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two‐dimensional [2D]) expansion. The system, based on the perfusion of bone marrow–nucleated cells through porous 3D scaffolds, supported the formation of stromal‐like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D‐expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.

Three-Dimensional Tissue Engineering of Hyaline Cartilage: Comparison of Adult Nasal and Articular Chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.

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.

Enzymatic Digestion of Adult Human Articular Cartilage Yields a Small Fraction of the Total Available Cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens ( n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.

Adult human neural crest-derived cells for articular cartilage repair.

HOX-negative, differentiated neural crest–derived adult cells from the nasal septum display self-renewal capacity and environmental plasticity and are compatible for articular cartilage repair. Cells from Nose Repair Tissue in Joint Cartilage repair remains a yet unmet clinical need, with few viable cell therapy options available. Taking cells from the knee or ankle to repair worn cartilage requires additional surgery and, in turn, pain and healing for the patient. As such, a new, accessible cell source would greatly benefit these patients. Here, Pelttari and colleagues looked up the nose for cells that may have the capacity to regenerate cartilage. Nasal septum cells arise from the neuroectoderm—the tissue that gives rise to the nervous system—and are better at repairing tissues than their mesoderm counterparts. These regenerative capabilities have been attributed to a lack of homeobox (HOX) gene expression. The authors therefore investigated whether nasal chondrocytes (HOX-negative, neuroectoderm origin) were compatible with an articular cartilage environment, like the knee joint (HOX-positive, mesoderm origin). The authors discovered that adult human nasal chondrocytes were able to self-renew and also, to their surprise, adopt a HOX-positive profile upon implantation into a mesoderm environment; in goats, this led to repair of experimental articular cartilage defects. In an ongoing clinical trial, human nasal chondrocytes have been shown to be safe once transplanted, suggesting translation of this new, easy-to-access cell source for repairing damaged joints. In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be distinguished from mesoderm-derived cells by their Hox-negative profile—a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differentiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also be reprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties of HOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autologous NC–based engineered tissues for the treatment of traumatic articular cartilage lesions.

Engineering human cell-based, functionally integrated osteochondral grafts by biological bonding of engineered cartilage tissues to bony scaffolds

In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone) using a fibrin gel (Tisseel). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and--since it is based on clinically compliant cells and materials--is amenable to be readily tested in the clinic.

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.

Perspective on the Evolution of Cell-Based Bone Tissue Engineering Strategies

Despite the compelling clinical needs in enhancing bone regeneration and the potential offered by the field of tissue engineering, the adoption of cell-based bone graft substitutes in clinical practice is limited to date. In fact, no study has yet convincingly demonstrated reproducible clinical performance of tissue-engineered implants and at least equivalent cost-effectiveness compared to the current treatment standards. Here, we propose and discuss how tissue engineering strategies could be evolved towards more efficient solutions, depicting three different experimental paradigms: (i) bioreactor-based production; (ii) intraoperative manufacturing, and (iii) developmental engineering. The described approaches reflect the need to streamline graft manufacturing processes while maintaining the potency of osteoprogenitors and recapitulating the sequence of biological steps occurring during bone development, including vascularization. The need to combine the assessment of efficacy of the different strategies with the understanding of their mechanisms of action in the target regenerative processes is highlighted. This will be crucial to identify the necessary and sufficient set of signals that need to be delivered at the injury or defect site and should thus form the basis to define release criteria for reproducibly effective engineered bone graft substitutes.