Robert Jan Kroeze

Adipose stem cells for intervertebral disc regeneration: current status and concepts for the future

•  Introduction •  Degenerative disc disease and emerging biological treatment approaches •  Stem cell sources •  Integration of ASC‐based regenerative medicine and surgery •  In vitro studies ‐  Animal models ‐  Cells in disc regeneration in vivo •  In vivo studies •  Perspective •  Conclusions

Reproducible long-term disc degeneration in a large animal model

Study Design. Twelve goats were chemically degenerated and the development of the degenerative signs was followed for 26 weeks to evaluate the progression of the induced degeneration. The results were also compared with a previous study to determine the reproducibility. Objectives. The purpose of this study was determine whether this Chondroitinase ABC (CABC) induced goat model is reproducible and to study the development of the degeneration in time up to 26 weeks. Summary of Background Data. Injecting CABC into goat intervertebral discs results in mild disc degeneration after 12 weeks. Spontaneous recovery or leveling off of the degeneration has been reported before and is relevant when the goat model is used in regeneration studies. Reproducibility of the induced degeneration is relevant as well. Methods. Twelve goats were used in this study. The development of degeneration was studied after the injection of 0.25 U/mL CABC intradiscally. The development of degenerative signs was studied after 18 (n = 6) and 26 (n = 6) weeks by means of radiograph, magnetic resonance imaging, macroscopic analysis, and histology and biochemical evaluation. The induced degeneration was compared with the results from a previous study, in which degeneration was induced similarly and analysis was performed after 12 weeks. Results. The severity of the degenerative signs was mild and was consequently present in all parameters analyzed. When compared with the results after 12 weeks, the degeneration was similar in the present study. Spontaneous recovery was not observed up to 26 weeks. Conclusion. The injection with CABC in the intervertebral disc reproducibly results in mild disc degeneration in the goat. These findings corroborate the goat model as a suitable large animal model to evaluate mild disc degeneration and potential new therapies.

Biodegradable Polymers in Bone Tissue Engineering

The use of degradable polymers in medicine largely started around the mid 20th century with their initial use as in vivo resorbing sutures. Thorough knowledge on this topic as been gained since then and the potential applications for these polymers were, and still are, rapidly expanding. After improving the properties of lactic acid-based polymers, these were no longer studied only from a scientific point of view, but also for their use in bone surgery in the 1990s. Unfortunately, after implanting these polymers, different foreign body reactions ranging from the presence of white blood cells to sterile sinuses with resorption of the original tissue were observed. This led to the misconception that degradable polymers would, in all cases, lead to inflammation and/or osteolysis at the implantation site. Nowadays, we have accumulated substantial knowledge on the issue of biocompatibility of biodegradable polymers and are able to tailor these polymers for specific applications and thereby strongly reduce the occurrence of adverse tissue reactions. However, the major issue of biofunctionality, when mechanical adaptation is taken into account, has hitherto been largely unrecognized. A thorough understanding of how to improve the biofunctionality, comprising biomechanical stability, but also visualization and sterilization of the material, together with the avoidance of fibrotic tissue formation and foreign body reactions, may greatly enhance the applicability and safety of degradable polymers in a wide area of tissue engineering applications. This review will address our current understanding of these biofunctionality factors, and will subsequently discuss the pitfalls remaining and potential solutions to solve these problems.

Rapid attachment of adipose stromal cells on resorbable polymeric scaffolds facilitates the one-step surgical procedure for cartilage and bone tissue engineering purposes

The stromal vascular fraction (SVF) of adipose tissue provides an abundant source of mesenchymal stem cells. For clinical application, it would be beneficial to establish treatments in which SVF is obtained, seeded onto a scaffold, and returned into the patient within a single surgical procedure. In this study, we evaluated the suitability of both a macroporous poly(L‐lactide‐co‐caprolactone) and a porous collagen type I/III scaffold for this purpose. Surprisingly, cell attachment was rapid (∼10 min) and sequestered the majority of adipose stem cells, as deduced from colony‐forming unit assays. Proliferation occurred in both polymeric scaffolds. Upon chondrogenic induction, up‐regulation of chondrogenic genes, production of glycosaminoglycans, and accumulation of collagen type II was observed, indicating differentiation of scaffold‐attached SVF cells along the chondrogenic lineage. Osteogenic differentiation was achieved in both scaffold types, as visualized by up‐regulation of osteogenic genes, increase of alkaline phosphatase production over time, and accumulation of bone sialoprotein and osteonectin. In conclusion, this study identifies both poly(L‐lactide‐co‐caprolactone) and collagen type I/III as promising scaffold materials for rapid attachment of adipose stem cell‐like (stromal) cells, enhancing the development of one‐step surgical concepts for cartilage and bone tissue engineering.

One-Step Surgical Procedure for the Treatment of Osteochondral Defects with Adipose-Derived Stem Cells in a Caprine Knee Defect: A Pilot Study

Abstract Regenerative therapies offer attractive alternatives for the treatment of osteochondral defects. Adipose-derived stromal vascular fraction (SVF) cells allow the development of one-step surgical procedures by their abundant availability and high frequency. In this pilot study we evaluated the in vivo safety, feasibility, and efficacy of this concept using scaffolds seeded with freshly isolated (SVF) or cultured adipose stem cells (ASCs), and compared these to their acellular counterparts. Osteochondral defects were created in medial condyles and trochlear grooves in knees of eight goats. Defects were filled with acellular collagen I/III scaffolds or scaffolds seeded with SVF cells or cultured ASCs. Osteochondral regeneration was evaluated after 1 and 4 months by macroscopy, immunohistochemistry, biomechanical analysis, microCT analysis, and biochemistry. After 1 month, no adverse effects were noted. Microscopic, but not macroscopic evaluation showed considerable yet not significant differences, with cell-loaded constructs showing more extensive regeneration. After 4 months, acellular constructs displayed increased regeneration, however, to a lesser degree than cell-treated constructs. The latter exhibited more extensive collagen type II, hyaline-like cartilage, and higher elastic moduli, and their glycosaminoglycan content in the cartilaginous layer better approached native tissue values. Moreover, their defect regions contained higher levels of regenerated, mature subchondral bone with more intense collagen type I staining. SVF cells tended to perform best on all parameters. In summary, this pilot study demonstrated the preclinical safety and feasibility of a one-step surgical procedure for osteochondral defect regeneration. Similar regeneration was found between freshly isolated SVF cells and cultured ASCs. Larger studies with longer follow-up are required to substantiate these findings.

Caprine articular, meniscus and intervertebral disc cartilage: an integral analysis of collagen network and chondrocytes

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.

Osteogenic differentiation strategies for adipose-derived mesenchymal stem cells.

Adipose stem cell preparations, either obtained as a freshly isolated so-called stromal vascular fraction (SVF) or as cells cultured to homogeneity and then referred to as adipose stem cells (ASCs), have found widespread use in a broad variety of studies on tissue engineering and regenerative medicine applications, including bone repair.For newcomers within the field, but also for established research laboratories having up to 10 years of expertise in this research area, it may be convenient to strive for, and use consensus protocols (1) for studying the osteogenic differentiation potential of ASC preparations in vitro, and (2) for osteogenic induction regimes for in vivo implementation. To assist in achieving this goal, this chapter describes various step-by-step osteogenic differentiation protocols for adipose-derived stem cell populations (SVF as well as ASCs) currently applied within our laboratory, with particular emphasis on protocols aimed at intra-operative use. The protocols describe the use of inducing compounds, including the bone morphogenetic proteins (BMPs), 1,25-dihydroxyvitamin-D3, and polyamines, as well as methods and parameters for evaluating the level of differentiation achieved.We would appreciate receiving feedback on the protocols described; this will facilitate the development of consensus protocols, which in turn will allow better comparison of data sets generated by different research groups. This continuing standardization, which might be reported on at international meetings like those of IFATS ( http://www.IFATS.org ), might be of benefit for the whole ASC research community.

The effect of ethylene oxide, glow discharge and electron beam on the surface characteristics of poly(L-lactide-co-caprolactone) and the corresponding cellular response of adipose stem cells

Bioabsorbable polymers are increasingly being used in tissue engineering strategies. Despite the knowledge that some sterilization techniques may affect the physical properties of these polymers, this aspect is often overlooked. We speculate that the type of sterilization method used may influence cellular responses by altering the surface characteristics. We cultured adipose stem cells on bioabsorbable poly(l-lactide-co-caprolactone) (PLCL) sheets, sterilized using either ethylene oxide (EO), argon glow discharge (aGD) or electron beam (e-beam). Significantly higher values for surface roughness in the order EO>aGD>e-beam and significant differences in contact angles (EO>e-beam>aGD) and surface energies (aGD>e-beam>EO) were observed. Increased cell attachment and proliferation rates were observed with lower contact angles. The alkaline phosphatase activity was significantly higher for the ethylene oxide sterilized PLCL sheet. In conclusion, the type of sterilization for bioabsorbable polymers should be considered in the design of new scaffolds, since it might affect, or can be used to enhance, the outcome of the tissue engineered construct.