The aim of the present study was to assess the influence of HDM_derm on two human primary cells – NHAC-kn and hMSC −, in terms of CPII, aggrecan, TGF-β1, IL-1β and MMP-3 release, in order to evaluate its possible use as a scaffold for in situ tissue engineering techniques in cartilage regenerative treatments. Human primary cells were chosen because they provide conditions that closely simulate a living model, thus yielding more physiologically significant results than animal-derived cells or cell lines. Since it is recognized that intra-species variability occurs, reproducibility of results was checked by taking into consideration dermis donor variability and not donor cell variability and thus four different dermis donors were tested.
The current results showed that HDM_derm supported NHAC-kn and hMSC cell adhesion, vitality, chondrogenic differentiation and synthetic activity. The NHAC-kn cultures maintained their phenotype with significantly increased synthesis of CPII and aggrecan, whereas the hMSC cultures showed a significant increase in aggrecan and TGF-β1 secretion. Furthermore, HDM-derm did not induce the release of IL-1β and MMP-3 that are often used as markers to measure inflammatory and catabolic stimuli in vitro. The comparison between NHAC-kn and hMSC normalized data revealed that, compared with hMSC, NHAC-kn were able to increase CPII synthesis at both experimental times, whereas hMSC were able to increase aggrecan at 7 days and TGF-β1 at both experimental times in comparison with NHAC-kn. In addition, the increased production of TGF-β1 by hMSC cells over the control wells may be responsible for the increased aggrecan synthesis via autocrine and paracrine pathways, as also suggested by other authors [15, 16]. In the present authors’ opinion, the differences observed between the cell-seeded construct and empty polystyrene wells were due to several factors: (1) stage of chondrogenic differentiation, NHAC-kn being already phenotypically differentiated chondrocyte cells; (2) the presence of a bioactive ECM membrane, as an environmental condition that is recognized as being a key factor in regulating cell behavior ; (3) the 3D-culture system condition, that is known to affect chondrogenesis in particular.
The reason why HDM_derm retained bioactivity upon decellularization might be because it participates and directly enhances cellular adhesion and proliferation indexes as found with NHAC-kn and hMSC, at least in the early stages of culture. Some authors found differences among growth factors (GFs) such as VEGF, FGF and TGF-β release in the extraction vehicle from different decellularized membranes [5, 18, 19]. Some TGF-β1 activity was still detected in the extract of the tested decellularized HDM_derm (428.36 ± 58.89 pg/mg) almost at the same concentration as the non-decellularized HDM_derm (417.01 ± 146.16 pg/mg), suggesting that HDM_derm is a bioactive substrate . In the present authors’ opinion, the native retained TGF-β1 is released from decellularized samples in a short culture time (within 6 days), thus affecting cell adhesion and early proliferation [5, 6, 8, 20]. After that, it might be assumed that the anabolic action of native TGF-β1 is almost completely exhausted by replacing medium twice a week and degradation processes, due to its finite lifespan.
Histological and SEM investigations supported in vitro data and showed that the HDM_derm was colonized by both cell types. Safranin O staining showed proteoglycans mainly at intracellular level and partially just around cells in both cultures, whereas Alcian Blue staining indicated glycosaminoglycan deposition at extracellular levels. These findings may suggest that, although GAGs have already been secreted by both primary cells, the macromolecules of proteoglycans, consisting of a core protein on which numerous GAG chains are attached, have yet to be assembled.
The experimental set up of the study had some weaknesses. First of all, the lack of a more clinically relevant matrix as a control was a major limitation. The ideal control material for the present study would be a dermis from the same donors decellularized by an already developed and recognized procedure. Unfortunately, these decellularization techniques that are used in the production of clinically accepted products are patented and it was not possible to reproduce this technique on the dermis derived from the donors included in the present study. Therefore, it was preferred to perform experiments without using other materials with different chemical and physical properties such as synthetic scaffolds or hydrogels.
The use of single-cell seeding density and relatively short culture time was the second weaknesses of the current experimental set up. Therefore, the seeding of HDM_derm probably needed an improvement in the in vitro culture conditions. In further in vitro experiments, the amount of cells seeded on the scaffold should be addressed, to try and increase the number up to 106 or 107 cells/ml, in order to study the influence of cell concentration on improving the building of a cartilage construct. However, the choice of cell density and experimental times was made to prevent irreversible contact-inhibition and senescence processes that affect NHAC-kn cell controls especially and in light of the authors’ previous in vitro experience with primary cultures of rat tenocytes seeded onto HDM_derm .
A further improvement in the current study set up might be to adopt a dynamic standardized culture condition by using bioreactors able to ensure efficient cell seeding, colonizing the scaffold deeply and also able to reproduce a mechanical stimulus to improve the cartilage-construct differentiation and maturation as well as regulate microenvironmental key factors in promoting chondrogenesis, such as oxygen concentration and pressure, or the presence of proteases [17, 21].
Finally, the strengths of the current study were: (1) the use of a new decellularization method on allogenic human dermis that provided a reliable 3D structural and biological scaffold for chondrocytes and MSCs; (2) the use of primary non-transformed human cells that closely simulate a living model, thus yielding more physiologically significant results than animal derived cells.
Various in vitro studies have been performed with different kinds of scaffolds with chondrocytes or MSCs for cartilage regeneration and most of them employed synthetic, biological or composite biomaterials such as poly-(lactic-co-glycolic acid) (PLGA), poly (lactic acid) PLA, poly (glycolic acid) PGA, hydrogel, chondroitin sulphate, hyaluronic acid, collagen, agarose, alginate, chitosan, gelatine, fibroin, fibrin glue or hybrid PLGA-gelatin/chondroitin sulphate/hyaluronic acid as cell supports [22–36].
Over the last few years, decellularized xenogenic and allogenic ECMs have started to be investigated for cartilage tissue engineering, because they retain structural and functional proteins and antibacterial activity, which has been shown within degradation products of biological scaffolds composed of extracellular matrix . However, the few studies testing ECMs, such as bovine cartilage , porcine adipose tissue , or cartilage [11, 13], human adipose tissue , or cartilage [9, 13], investigating mainly the behaviour of chondrocytes or MSCs in terms of proliferation and viability, concluded that ECMs provided suitable 3D substrates not only for the growth of cells, but also for promoting the formation of new cartilaginous engineered tissue. To our knowledge, the current study is the first to address the use of human decellularized ECM as an alternative 3D scaffold for cartilage regeneration. By comparing the present results to previously cited literature [8–13], it is evident that inflammatory stimuli, and anabolic and catabolic synthetic activity are not deeply investigated biological aspects to evaluate a possible influence of ECM on chondrocyte or MSC behaviour. The decellularization technique employed for the present scaffold did not adversely affect the structural integrity and biological activity of the remaining ECM of HDM_derm, thus its mechanical competence was maintained [6, 14]. The advantage of the technique, consisting of a combination of trypsin washes and the extremely low dosage of gamma-ray irradiation (about 0.1 kGy), is to avoid the use of strong chemical agents, which might lead to toxic leachables in the decellularized products, and the terminal sterilization process, which are known to be the main causes of distinct host tissue histological and morphologic responses [6, 14].
Further in vitro studies are mandatory to test the use of HDM_derm with tissue engineering techniques. The present study on the possible application of HDM_derm for cartilage suggests that this derived matrix might be investigated also as a functional scaffold for chondrocyte repopulation for in situ engineering techniques. HDM_derm seems to be a suitable matrix for cartilage regeneration, because it has been shown by the present study to maintain cellular viability and differentiation, and to retain structure and bioactivity, particularly TGF-β1 that might counteract catabolic IL-1β effects, whose levels have been found to correlate with the severity of cartilage damage in vivo. Finally, all in vitro data need to be transferred in vivo into specific preclinical validated models of acute, chronic or degenerative cartilage lesions to assess the therapeutic and functional effectiveness of HDM_derm in cartilage repair or regeneration.