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Evaluation of different commercial hyaluronic acids as a vehicle for injection of human adipose-derived mesenchymal stem cells 1 Camila Cohen Kaleka and Eder Zucconi contributed equally to this study and share the authorship as the first authors of this scientific research.

ABSTRACT

Objective:

The main purpose of this study is to evaluate, in vitro, the cytotoxicity of different commercial brands of hyaluronic acids to be used as a vehicle for injection of human adipose-derived mesenchymal stem cells (AD-MSCs).

Methods:

AD-MSCs were divided into seven groups: one control group where AD-MSCs were cultivated with phosphate-buffered saline (PBS) and six other groups where AD-MSCs were cultivated with different commercial brands of hyaluronic acid. AD-MSC viability analysis was performed after 4, 24, and 48 h in contact with each treatment, using the trypan staining method on a Countess automated cell counter (Thermo Fisher Scientific).

Results:

The results clearly demonstrated a significant difference in cell viability when AD-MSCs were exposed to different hyaluronic acids when compared with the control group.

Conclusion:

These data suggest that hyaluronic acid can be used as a vehicle for injection of human AD-MSCs, but caution is needed to choose the best product, aiming at its future therapeutic application.

Keywords:
Cartilage diseases; Knee; Arthroscopy; Articular cartilage; Mesenchymal stem cells; Mesenchymal stem cell transplantation

RESUMO

Objetivo:

Avaliar in vitro, de forma direta, a citotoxicidade de ácidos hialurônicos como veículo de injeção para linhagens de células-tronco mesenquimais (CTMs) obtidas de tecido adiposo humano.

Métodos:

As CTMs foram divididas em sete grupos, os quais foram expostos ao ácido hialurônico de seis marcas comerciais, além do contato com tampão fosfato-salino PBS (grupo controle). Após quatro, 24 e 48 horas, foi feita a análise da viabilidade celular através do contador Countess pelo método de coloração com Trypan Blue (Thermo Fisher Scientific).

Resultados:

Os resultados demonstraram uma diferença significativa na viabilidade celular quando essas linhagens de CTMs foram expostas aos diferentes ácidos hialurônicos em comparação com o grupo controle.

Conclusão:

Os dados sugerem que o ácido hialurônico pode ser usado como veículo de injeção para CTMs, porém é necessária cautela na escolha do melhor produto para aplicação terapêutica futura.

Palavras-chave:
Doenças da cartilagem; Joelho; Artroscopia; Cartilagem articular; Células-tronco mesenquimais; Transplante de células-tronco mesenquimais

Introduction

Articular cartilage lesions are among the most frequent musculoskeletal pathologies, and the most difficult to treat. This is due to the fact that the chondral tissue is subject to constant stimuli; moreover, it has a low potential for repair due to its vascular and lymphatic circulation deficiency.11 Oshima Y, Harwood FL, Coutts RD, Kubo T, Amiel D. Variation of mesenchymal cells in polylactic acid scaffold in an osteochondral repair model. Tissue Eng Part C Methods. 2009;15(4):595-604.,22 Wescoe KE, Schugar RC, Chu CR, Deasy BM. The role of the biochemical and biophysical environment in chondrogenic stem cell differentiation assays and cartilage tissue engineering. Cell Biochem Biophys. 2008;52(2):85-102.

Among the currently available surgical treatment options, one that presents encouraging results is the autologous chondrocyte implant described in 1994 by Brittberg et al., in which, after collection of healthy cartilage, cell expansion and culture are performed and, at a second time, the chondrocytes are implanted in the chondral lesion.33 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994;331(14):889-95.,44 Yasui Y, Ando W, Shimomura K, Koizumi K, Ryota C, Hamamoto S, et al. Scaffold-free, stem cell-based cartilage repair. J Clin Orthop Trauma. 2016;7(3):157-63. Due to the limitations for this procedure, such as unavailability and degeneration of donor cartilage, problems with in vitro chondrocyte expansion, and its high cost, viable optional forms have been sought to treat this important joint lesion.11 Oshima Y, Harwood FL, Coutts RD, Kubo T, Amiel D. Variation of mesenchymal cells in polylactic acid scaffold in an osteochondral repair model. Tissue Eng Part C Methods. 2009;15(4):595-604.,55 Filardo G, Madry H, Jelic M, Roffi A, Cucchiarini M, Kon E. Mesenchymal stem cells for the treatment of cartilage lesions: from preclinical findings to clinical application in orthopaedics. Knee Surg Sports Traum Arthrosc. 2013;21(8):1717-29. Thus, mesenchymal stem cells (MSCs) have been identified as a good treatment option. Among the various sources of production, adipose-tissue derived MSCs (AD-MSCs) are highlighted, as they present potentiality for regeneration and differentiation in cartilaginous tissue, are available in large amounts in the body and can be obtained with easy and non-invasive techniques.66 Açil Y, Zhang X, Nitsche T, Möller B, Gassling V, Wiltfang J, et al. Effects of different scaffolds on rat adipose tissue derived stroma cells. J Craniomaxillofac Surg. 2014;42(6):825-34.

7 Gimble JM, Guilak F, Bunneli BA. Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells. Stem Cell Res Ther. 2010;1(2):19.
-88 Kim HJ, Im GI. Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: greater doses of growth factor are necessary. J Orthop Res. 2009;27(5):612-9. Hyaluronic acid is considered an excellent vehicle for MSCs in tissue repair, due to its viscosity and physicochemical properties, and it has gained prominence in tissue bioengineering99 Gojgini S, Tokatlian T, Segura T. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels. Mol Pharm. 2011;8(5):1582-91.,1010 Lam J, Truong NF, Segura T. Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds. Acta Biomater. 2014;10(4):1571-80. (Table 1). Nonetheless, no data assessing the potential of MSCs when exposed to different hyaluronate molecular weights, brands, and viscosities are available.

Table 1
Physical and chemical properties of different hyaluronic acids.

This study is aimed at evaluating in vitro, in a direct way, the cytotoxicity of different brands of hyaluronic acid on MSC lineages obtained from human adipose tissue.

Material and methods

Ethical considerations

All human tissue samples for MSC isolation were obtained after the informed consent form was signed by the donor or guardian, in agreement with the Research in Human Beings Ethics Committee of the University Hospital and Institute of Biosciences of the University of São Paulo (protocol no. 040/2005).

Collection, isolation, and expansion of MSCs

AD-MSCs were isolated using methods previously described by the present group.1111 Vieira NM, Bueno CR, Brandalise V, Moraes LV, Zucconi E, Secco M, et al. SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression. Stem Cells. 2008;26(9):2391-8.

12 Vieira NM, Zucconi E, Bueno CR, Secco M, Suzuki MF, Bartolini P, et al. Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice. Stem Cell Rev. 2010;6(4):560-6.

13 Secco M, Zucconi E, Vieira NM, Fogaça LL, Cerqueira A, Carvalho MD, et al. Multipotent stem cells from umbilical cord: cord is richer than blood!. Stem Cells. 2008;26(1):146-50.
-1414 Vieira NM, Brandalise V, Zucconi E, Jazedje T, Secco M, Nunes VA, et al. Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro. Biol Cell. 2008;100(4):231-41. Adipose tissue was collected in cesarean sections from the abdominal subcutaneous region of the patient. After collection, the samples were stored in a sterile flask and transported to the laboratory in thermal boxes with temperature control between 4 °C and 24 °C. All samples were processed within 48 h.

Briefly, to isolate the AD-MSCs, the adipose tissue samples were washed in phosphate-buffered saline (PBS) 1x, pH 7.4, with 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies), and then dissociated with 0.075% GMP (Serva) collagenase at 37 °C for 30 min. Then, the infranatant was centrifuged at 300 × g for 5 min, and the cell pellet formed was plated on culture bottles, following a ratio of 1000-3500 cells per cm2.

For in vitro expansion of the AD-MSCs, the animal serum-free MSC culture system StemPro MSC SFM (Thermo Fisher Scientific) was used according to the supplier's recommendations (proliferation medium). Each lineage was maintained at 37 °C in a humidified atmosphere with 5% CO2 until reaching at least 1 × 106 cells to be cryopreserved in liquid nitrogen at −196 °C with the StemPro MSC SFM kit. To achieve the number of cells desirable for the experiments, they were thawed in a 37 °C water bath and plated in culture bottles with the abovementioned proliferation medium.

Immunophenotyping

In order to confirm the mesenchymal origin of the obtained AD-MSC lineages, cell membrane protein expression analysis was performed by flow cytometry, using the methods previously described by the present group.1111 Vieira NM, Bueno CR, Brandalise V, Moraes LV, Zucconi E, Secco M, et al. SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression. Stem Cells. 2008;26(9):2391-8.

12 Vieira NM, Zucconi E, Bueno CR, Secco M, Suzuki MF, Bartolini P, et al. Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice. Stem Cell Rev. 2010;6(4):560-6.

13 Secco M, Zucconi E, Vieira NM, Fogaça LL, Cerqueira A, Carvalho MD, et al. Multipotent stem cells from umbilical cord: cord is richer than blood!. Stem Cells. 2008;26(1):146-50.
-1414 Vieira NM, Brandalise V, Zucconi E, Jazedje T, Secco M, Nunes VA, et al. Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro. Biol Cell. 2008;100(4):231-41. To that end, the adherent cells obtained in stage five were incubated with the following conjugated antibodies: CD29-PE-Cy5, CD31-PE, CD34-PerCP, CD45-FITC, CD73-PE, CD90-R-PE, CD105-PE, HLA-ABC-FITC, HLA-DR-R-PE (Becton Dickinson), according to the manufacturer's recommendations. As a negative control, cells were incubated with PBS instead of the primary antibody. A total of 10,000 events were acquired with the Guava EasyCyte flow cytometer (Guava Technologies) and analyzed with Guava ExpressPro software (Guava Technologies).

Differentiation test

hASC and hUCT were submitted to differentiation protocols to evaluate their MSC properties.1313 Secco M, Zucconi E, Vieira NM, Fogaça LL, Cerqueira A, Carvalho MD, et al. Multipotent stem cells from umbilical cord: cord is richer than blood!. Stem Cells. 2008;26(1):146-50.,1414 Vieira NM, Brandalise V, Zucconi E, Jazedje T, Secco M, Nunes VA, et al. Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro. Biol Cell. 2008;100(4):231-41. Thus, the adherent cells obtained in stage 5 were submitted to adipogenic, chondrogenic, and osteogenic in vitro inductions with the StemPro differentiation kit (Life Technologies) according to the manufacturer's recommendations. Non-induced control samples were maintained in proliferation medium throughout the assay.

Adipogenesis

Adipogenic differentiation was confirmed on day 21 by staining with Oil Red O (Sigma). For this, the cells were set with 4% paraformaldehyde for 30 min, washed with distilled water, and stained with a working solution of 0.16% Oil Red O for 20 min.

Chondrogenesis

To confirm chondrogenic differentiation, after 21 days of induction the cells were stained with 1% toluidine blue for 1 min, washed with distilled water, and then treated with a wash of 70%, 95%, and 100% ethyl alcohol solution, respectively.

Osteogenesis

Osteogenic differentiation was assessed by the Von Kossa stain. Briefly, the cells were stained with 1% silver nitrate (Sigma) for 45 min under ultraviolet light, stained with 3% sodium thiosulfate (Sigma) for 5 min, and then contrasted with van Gieson. At the end of this step, the cells were carefully washed with ethyl alcohol and allowed to dry completely.

Experimental groups

In step eight, MSCs were divided into seven groups, which were exposed to hyaluronic acid from six commercial brands and phosphate-saline buffer - PBS (control group):

  • Group 1: AD-MSC in contact with Fermathron® (Hyaltech),

  • Group 2 AD-MSC in contact with Orthovisc® (J&J),

  • Group 3 AD-MSC in contact with Synvisc® (Sanofi),

  • Group 4 AD-MSC in contact with Synovium® (LCA Pharmaceutical),

  • Group 5 AD-MSC in contact with Suprahyal® (Zodiac),

  • Group 6 AD-MSC in contact with Osteonil® (TRB Chemedica),

  • Group 7 AD-MSC in contact with PBS (Thermo Fisher Scientific).

In the preparation process, the cells were detached from the culture bottle with TrypLE reagent (Thermo Scientific) and centrifuged at 300 × g for 5 min, counted in the Countess apparatus (Thermo Fisher), and resuspended in hyaluronic acid or PBS at the concentration of. After 4, 24, and 48 h of incubation at room temperature and atmosphere, cell viability analysis was performed through the Countess counter by the Trypan Blue staining method (Thermo Fisher Scientific), according to the supplier's recommendations. Two independent experiments were performed, each with three different AD-MSC strains (AT8, AT11, and AT13). For each time interval, a single viability reading was made, and the same experimental conditions were followed.

Statistical analysis

The numerical data obtained were presented as a mean ± standard deviation. Statistical analysis was performed using Student's t-test (p ≤ 0.05).

Results

Establishment and selection of cell lineages

Three AD-MSC lineages were obtained: AT8, AT11, and AT13. They were followed in vitro, and the morphology of each of them was evaluated during the stages. All lineages presented morphology similar to MSCs (Fig. 1) and excellent proliferation rate in the stages evaluated, in an animal serum-free medium. Therefore, they are excellent candidates for cell expansion protocols for future therapeutic applications.

Fig. 1
Establishment of AD-MSC lineages. Morphology of adherent cells obtained from a human adipose tissue sample (AT8, AT11, and AT13). The photos represent the in vitro cultures in stage eight. 50× magnification.

Immunophenotyping

To confirm the mesenchymal origin of the adherent cell lineages obtained (AT8, AT11, and AT13), the authors used flow cytometry to evaluate the expression of a panel of cell membrane proteins. Samples were positive for CD29 and CD90 adherent cell markers; negative for the CD31 endothelial marker; negative for the CD34 and CD45 hematopoietic markers; positive for the CD73 and CD105 mesenchymal markers; positive for HLA-ABC; and negative for HLA-DR (Fig. 2). This pattern is in accordance with the immunophenotyping panel set forth by the International Society of Cell Therapy.1515 Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7.

Fig. 2
Immunophenotyping of adherent cells obtained from adipose tissue. Gray histograms represent the population of non-antibody labeled cells (negative control). Black histograms represent the population of cells labeled with the respective antibodies described in each line. The graphs show the number of cells versus fluorescence intensity.

Differentiation test

The plasticity of adipose cells obtained from adipose tissue (lineages AT8, AT11, and AT13) was evaluated after 21 days of culture in a medium that induces specialized cell types of mesodermal origin. The osteogenic (Fig. 3A), adipogenic (Fig. 3B), and chondrogenic (Fig. 3C) inductions were confirmed by the presence of calcium deposits, lipid vacuoles, and extracellular matrix rich in mucopolysaccharides, respectively. Together, these results indicate the mesenchymal origin of isolated adherent cells and the potential for multipotent in vitro differentiation.1515 Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7.

Fig. 3
The potential for differentiation of adherent cells isolated from adipose tissue. The first column represents the negative control lineages (not induced) and the second column, the lineages treated with induction medium after 21 days of culture. (A) Osteogenic induction. Differentiation was confirmed by the presence of calcium deposits (dark brown) through von Kossa staining. 50× magnification. (B) Adipogenesis induction. The differentiation was confirmed by the presence of droplets of cytoplasmic lipids (red) by Oil Red O staining. 200× magnification. (C) Chondrogenic induction. The differentiation was confirmed by toluidine blue staining, which allows visualizing the phenomenon of metachromasia, characteristic of extracellular matrix rich in mucopolysaccharides. 50× magnification.

Hyaluronic acid contact test

The results found in the study indicated a difference in cell viability according to the hyaluronic acid used to culture the MSCs in comparison with the control group, as shown in Table 2.

Table 2
Viability of adipose tissue-derived human mesenchymal stem cells in contact with different commercial hyaluronic acids. Mean and standard deviation expressed as a percentage of viability for each treatment evaluated.

In the first 4 h, only the cells in contact with Orthovisc® presented a significantly reduced viability in relation to the control group. The same effect was observed in the cells in contact with Orthovisc® for 24 h and 48 h (p < 0.05). However, after 24 h, all treatments, except for the cells in contact with Suprahyal®, had significantly reduced viability when compared with the PBS control group. Finally, after 48 h, both cells in contact with Orthovisc® and Synvisc® had significantly reduced viability when compared with the PBS control group.

Thus, Suprahyal® was the most suitable hyaluronic acid for use as a vehicle for AD-MSC transport in cell therapy trials, maintaining the viability at levels very close to that of the cells treated with the PBS control solution.

Regarding viscosity and cell manipulation, the hyaluronic acids Fermathron® and Suprahyal® presented the best performance. Synovium® acid presented very high viscosity, which hindered cell manipulation. In turn, Orthovisc® and Synvisc® presented intermediate viscosity, but also hindered cell manipulation.

Discussion

Regenerative medicine has been viewed with great interest by orthopedists, as it has become an attractive option for the treatment of chronic conditions, such as joint cartilage lesions and arthritis. This is due to the fact that the treatments available to date are insufficient for complete lesion healing, which often results in decreased mobility and loss of patient autonomy. This tissue regeneration therapy uses cell therapy as a strategy, in which stem cells are used for lesion treatment and repair. Thus, due to their potential differentiation capacity in bone and cartilaginous tissue, MSCs are very promising and can be isolated from several sites in the human organism, such as bone marrow, fat, periosteum, and synovial membrane, among others.1616 Schmitt A, Van Griensven M, Imhoff AB, Buchmann S. Application of stem cells in orthopedics. Stem Cells Int. 2012;:394962.,1717 Ehnert S, Glanemann M, Schmitt A, Vogt S, Shanny N, Nussler NC, et al. The possible use of stem cells in regenerative medicine: dream or reality?. Langenbecks Arch Surg. 2009;394(6):985-97.

Adipose tissue is considered a good candidate as a source for cell therapy because it can be accessed by relatively simple and non-invasive techniques and has a high concentration of MSCs, approximately 10-100 times more MSCs per volume than bone marrow.1818 Gimble JM, Grayson W, Guilak F, Lopez MJ, Vunjak-Novakovic G. Adipose tissue as a stem cell source for musculoskeletal regeneration. Front Biosci (Schol Ed). 2011;3:69-81.

However, isolated cell therapy is not yet capable of repairing large defects. In this sense, tissue engineering is seen as a promising strategy, as it uses scaffolds as biomaterials to mimic the extracellular matrix architecture of the specific tissue and has a great influence on cell growth and differentiation.1717 Ehnert S, Glanemann M, Schmitt A, Vogt S, Shanny N, Nussler NC, et al. The possible use of stem cells in regenerative medicine: dream or reality?. Langenbecks Arch Surg. 2009;394(6):985-97. This fact has already been observed by Hidaka et al.,1919 Hidaka M, Su GN, Chen JK, Mukaisho K, Hattori T, Yamamoto G. Transplantation of engineered bone tissue using a rotary three-dimensional culture system. In Vitro Cell Dev Biol Anim. 2007;43(2):49-58. He et al.,2020 He L, Nan X, Wang Y, Guan L, Bai C, Shi S, et al. Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells. Sci China C Life Sci. 2007;50(4):429-37. and Marra et al.2121 Marra KG, Defail AJ, Clavijo-Alvarez JA, Badylak SF, Taieb A, Schipper B, et al. FGF-2 enhances vascularization for adipose tissue engineering. Plast Reconstr Surg. 2008;121(4):1153-64. in 2007 and 2008, when successful results were observed in the use of scaffolds with progenitor cells to replace bone, cutaneous, and adipose tissue, respectively.

Hyaluronic acid is commonly used in tissue engineering as a biomaterial in hydrogel structures. Therefore, understanding its functions is fundamental for creating more efficient structures.1010 Lam J, Truong NF, Segura T. Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds. Acta Biomater. 2014;10(4):1571-80.

One of the explanations for the superior results of the PBS is due to its isotonic nature, which makes this the most commonly used reagent in biological research. Phosphate salts are non-toxic to living cells and have a high buffering capacity. pH maintenance is important for cell viability, as well as in the process of recovering and revitalizing cells. In turn, hyaluronic acid is one of the main components of the extracellular matrix and significantly contributes to cell proliferation and migration.

Based on the present results, it was observed that the cell viability of the AD-MSCs is strongly associated with the type of hyaluronic acid used for culture. Hyaluronic acid is one of the main components of the extracellular matrix and significantly contributes to cell proliferation and migration.

Furthermore, it is believed that the molecular weight may be responsible for the differences observed. Among the brands researched, Suprahyal® presented the highest amount of viable stem cells at the end of the analyzed period and the lowest molecular weight (0.75 MDa) among the evaluated groups. This can be explained by the evidence that high molecular weight hyaluronic acid acts as an inhibitor of angiogenesis and cell proliferation, in addition to having an anti-inflammatory and immunosuppressive effect.1010 Lam J, Truong NF, Segura T. Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds. Acta Biomater. 2014;10(4):1571-80.

Therefore, it can be suggested that cell viability may be directly related to the concentration and molecular weight of the hyaluronic acid used and that this may be a determining factor in the success of the surgical treatment of chondral lesions.

However, it is important to note that the abovementioned results regard the influence of hyaluronic acid on stem cells in the context of tissue engineering and not their use to provide viscosupplementation in the conservative treatment of osteoarthritis; for the latter purpose, there is evidence of the clinical benefit and the probable superiority of high molecular weight hyaluronic acid.2222 Ricci M, Micheloni GM, Berti M, Perusi F, Sambugaro E, Vecchini E, et al. Clinical comparison of oral administration and viscosupplementation of hyaluronic acid (HA) in early knee osteoarthritis. Musculoskelet Surg. 2016 [Epub ahead of print] PubMed PMID: 27681813.

23 Conrozier T, Bozgan AM, Bossert M, Sondag M, Lohse-Walliser A, Balblanc JC. Standardized follow-up of pacientes with symptomatic knee osteoarthritis treated with a single intra-articular injection of a combination of cross-linked hyaluronic acid and mannitol. Clin Med Insights Arthritis Musculoskelet Disord. 2016;9:175-9.
-2424 Maheu E, Rannou F, Reginster JY. Efficacy and safety of hyaluronic acid in the management of osteoarthritis: evidence from real-life setting trials and surveys. Semin Arthritis Rheum. 2016;45(4 Suppl):S28-33.

Conclusion

The data suggest that hyaluronic acid can be used as an injection vehicle for MSCs, but caution is needed in the choice of the best product according to the viability and cytotoxic effect of the different brands of hyaluronic acids. Thereby, the therapeutic application will be performed more safely.

  • Study conducted at Laboratório StemCorp de Tecnologia em células-tronco, together with the Instituto Cohen de Ortopedia, Reabilitação e Medicina do Esporte, São Paulo, SP, Brazil.

Acknowledgements

Thanks go to Dr Mayana Zatz of the Center for Research on the Human Genome and Stem Cells (IB-USP), who kindly allowed the use of her laboratory's culture room and equipment. This work was financed with funding from the National Council for Scientific and Technological Development (CNPq).

References

  • 1
    Oshima Y, Harwood FL, Coutts RD, Kubo T, Amiel D. Variation of mesenchymal cells in polylactic acid scaffold in an osteochondral repair model. Tissue Eng Part C Methods. 2009;15(4):595-604.
  • 2
    Wescoe KE, Schugar RC, Chu CR, Deasy BM. The role of the biochemical and biophysical environment in chondrogenic stem cell differentiation assays and cartilage tissue engineering. Cell Biochem Biophys. 2008;52(2):85-102.
  • 3
    Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994;331(14):889-95.
  • 4
    Yasui Y, Ando W, Shimomura K, Koizumi K, Ryota C, Hamamoto S, et al. Scaffold-free, stem cell-based cartilage repair. J Clin Orthop Trauma. 2016;7(3):157-63.
  • 5
    Filardo G, Madry H, Jelic M, Roffi A, Cucchiarini M, Kon E. Mesenchymal stem cells for the treatment of cartilage lesions: from preclinical findings to clinical application in orthopaedics. Knee Surg Sports Traum Arthrosc. 2013;21(8):1717-29.
  • 6
    Açil Y, Zhang X, Nitsche T, Möller B, Gassling V, Wiltfang J, et al. Effects of different scaffolds on rat adipose tissue derived stroma cells. J Craniomaxillofac Surg. 2014;42(6):825-34.
  • 7
    Gimble JM, Guilak F, Bunneli BA. Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells. Stem Cell Res Ther. 2010;1(2):19.
  • 8
    Kim HJ, Im GI. Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: greater doses of growth factor are necessary. J Orthop Res. 2009;27(5):612-9.
  • 9
    Gojgini S, Tokatlian T, Segura T. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels. Mol Pharm. 2011;8(5):1582-91.
  • 10
    Lam J, Truong NF, Segura T. Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds. Acta Biomater. 2014;10(4):1571-80.
  • 11
    Vieira NM, Bueno CR, Brandalise V, Moraes LV, Zucconi E, Secco M, et al. SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression. Stem Cells. 2008;26(9):2391-8.
  • 12
    Vieira NM, Zucconi E, Bueno CR, Secco M, Suzuki MF, Bartolini P, et al. Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice. Stem Cell Rev. 2010;6(4):560-6.
  • 13
    Secco M, Zucconi E, Vieira NM, Fogaça LL, Cerqueira A, Carvalho MD, et al. Multipotent stem cells from umbilical cord: cord is richer than blood!. Stem Cells. 2008;26(1):146-50.
  • 14
    Vieira NM, Brandalise V, Zucconi E, Jazedje T, Secco M, Nunes VA, et al. Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro. Biol Cell. 2008;100(4):231-41.
  • 15
    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7.
  • 16
    Schmitt A, Van Griensven M, Imhoff AB, Buchmann S. Application of stem cells in orthopedics. Stem Cells Int. 2012;:394962.
  • 17
    Ehnert S, Glanemann M, Schmitt A, Vogt S, Shanny N, Nussler NC, et al. The possible use of stem cells in regenerative medicine: dream or reality?. Langenbecks Arch Surg. 2009;394(6):985-97.
  • 18
    Gimble JM, Grayson W, Guilak F, Lopez MJ, Vunjak-Novakovic G. Adipose tissue as a stem cell source for musculoskeletal regeneration. Front Biosci (Schol Ed). 2011;3:69-81.
  • 19
    Hidaka M, Su GN, Chen JK, Mukaisho K, Hattori T, Yamamoto G. Transplantation of engineered bone tissue using a rotary three-dimensional culture system. In Vitro Cell Dev Biol Anim. 2007;43(2):49-58.
  • 20
    He L, Nan X, Wang Y, Guan L, Bai C, Shi S, et al. Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells. Sci China C Life Sci. 2007;50(4):429-37.
  • 21
    Marra KG, Defail AJ, Clavijo-Alvarez JA, Badylak SF, Taieb A, Schipper B, et al. FGF-2 enhances vascularization for adipose tissue engineering. Plast Reconstr Surg. 2008;121(4):1153-64.
  • 22
    Ricci M, Micheloni GM, Berti M, Perusi F, Sambugaro E, Vecchini E, et al. Clinical comparison of oral administration and viscosupplementation of hyaluronic acid (HA) in early knee osteoarthritis. Musculoskelet Surg. 2016 [Epub ahead of print] PubMed PMID: 27681813.
  • 23
    Conrozier T, Bozgan AM, Bossert M, Sondag M, Lohse-Walliser A, Balblanc JC. Standardized follow-up of pacientes with symptomatic knee osteoarthritis treated with a single intra-articular injection of a combination of cross-linked hyaluronic acid and mannitol. Clin Med Insights Arthritis Musculoskelet Disord. 2016;9:175-9.
  • 24
    Maheu E, Rannou F, Reginster JY. Efficacy and safety of hyaluronic acid in the management of osteoarthritis: evidence from real-life setting trials and surveys. Semin Arthritis Rheum. 2016;45(4 Suppl):S28-33.

Publication Dates

  • Publication in this collection
    Sep-Oct 2018

History

  • Received
    15 Jan 2017
  • Accepted
    19 May 2017
  • Published
    2 Aug 2018
Sociedade Brasileira de Ortopedia e Traumatologia Al. Lorena, 427 14º andar, 01424-000 São Paulo - SP - Brasil, Tel.: 55 11 2137-5400 - São Paulo - SP - Brazil
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