Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV were observed. After 56 days in culture, the bioprinted constructs were still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results showed a promising procedure to obtain 3D cartilage-like constructs that could be potential use as stable chondral tissue implants for future therapies.
Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells that could potentially be used to repair injured cartilage in diseases. The objetive was to analyze different sources of human MSCs to find a suitable alternative source for the isolation of MSCs with high chondrogenic potential. Femoral bone marrow, adipose tissue from articular and subcutaneous locations (hip, knee, hand, ankle and elbow) were obtained from 35 patients who undewent different types of orthopedic surgery (21 women, mean age 69.83 ± 13.93 (range 38–91) years. Neoplasic and immunocompromised patients were refused. The Ethical Committee for Clinical Research of the Government of Aragón (CEICA) approved the study and all patients provided informed consent. Cells were conjugated wiith monoclonal antibodies. Cell fluorescence was evaluated by flow cytometry using a FACSCalibur flow cytometer and analysed using CellQuest software (Becton Dickinson). Chondrogenic differentiation of human MSCs from the various tissues at P1 and P3 was induced in a 30-day micropellet culture [Pittenger et al., 1999]. To evaluate the differentiation of cartilaginous pellet cultures, samples were fixed embedded in paraffin and cut into 5- υm-thick slices. The slices were treated with hematoxylin-eosin and safranin O (Sigma-Aldrich). Each sample was graded according to the Bern Histological Grading Scale [Grogan et al., 2006], which is a visual scale that incorporates three parameters indicative of cartilage quality: uniform and dark staining with safranin O, cell density or extent of matrix produced and cellular morphology (overall score 0–9). Stained sections were evaluated and graded by two different researchers under a BX41 dual viewer microscope or a Nikon TE2000-E inverted microscope with the NIS-Elements software. Statistics were calculated using bivariate analysis. Pearson's χ2 or Fisher's exact tests were used to compare the Bern Scores of various tissues. To evaluate the cell proliferation, surface marker expression and tissue type results, ANOVA or Kruskal-Wallis tests were used, depending on the data distribution. Results were considered to be significant when p was < 0.05. MSCs from all tissues analysed had a fibroblastic morphology, but their rates of proliferation varied. Subcutaneous fat derived MSCs proliferated faster than bone marrow. MSCs from Hoffa fat, hip and knee subcutaneous proliferated slower than MSCs from elbow, ankle and hand subcutaneous. Flow cytometry: most of cells lacked expression of CD31, CD34, CD36, CD117 (c-kit), CD133/1 and HLA-DR. At same time 95% of cells expressed CD13, CD44, CD59, CD73, CD90, CD105, CD151 y CD166. Fenotype showed no differences in cells from different anatomic places. Cells from hip and knee subcutaneous showed a worst differentiation to hyaline cartilage. Hoffa fat cells showed high capacity in transforming to hyaline cartilage. Cells from different anatomic places show different chondrogenic potential that has to be considered to choose the cells source.
Human cells: CD13+ (94–99%), CD44+ (87–99), CD49d (14–70%), CD90+ (92–99%), CD105+ (90–97%), CD 117-BD+ (2–22%). Sheep cells presented CD13+ (32–70%), CD34-, CD36, CD44+ (90–96%), CD49d (40–80%), CD54+ (50–80%), CD90+ (90–97%), CD105+ (10–25%). CD117-BD+ (48–76%). Rabbits cells: CD13+ (14–78%), CD44+ (10–80%), CD49d (2–9%), CD90+ (27–92%), CD105+ (2–24%), CD 117-BD+ (15–57%). Human cells number/mL did not show significant differences between patients, or between P0 0 (14 culture days) (average mean: 525000 ± 298956) and P5 (525000), nevertheless the average mean decreased from P5 to P6 (130.000) until P8 (111 culture days) (85.000). Rabbits cells number/mL did not show significant differences between P0 (673000 ± 379697) and P1 (596000 ± 488740) and decreased in P2 (299500 ± 159161) without any significant change in P8. Ovine cells number/mL average mean in P0 was 1.370.600 (± 802758), this decreased in P1 (420000 ± 95197) however, showed no significant changes in P8 (291875 ± 86394).
Female sex and a greater frequency of hallux rigidus have a statistically significant relationship (p=0.095) for a 90% confidence interval. This is contrary to the opinion expressed in most of the literature published up to the present. The height of the patient and a greater frequency of hallux rigidus seem to have a statistically significant relationship for a 90% confidence interval (p=0.067). This has not been mentioned up to now in any published paper. The For factors such as hypermobility of the 1st toe, excess of the 1st axis, adductor metatarsus, interphalangeal hallux valgus and chevron shaped joint, we have found no significant relationship with the development of hallux rigidus.
