Comparison of articular cartilage repair with different hydrogel-human umbilical cord blood-derived mesenchymal stem cell composites in a rat model
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The present work was designed to explore the feasibility and efficacy of articular cartilage repair using composites of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) and four different hydrogels in a rat model.
Full-thickness articular cartilage defects were created at the trochlear groove of femur in both knees of rats. Composites of hUCB-MSCs and four different hydrogels (group A, 4% hyaluronic acid; group B, 3% alginate:30% pluronic (1:1, v/v); group C, 4% hyaluronic acid: 3% alginate: 20% pluronic (2:1:1, v/v}; and group D, 4% hyaluronic acid:3% alginate:20% pluronic;chitosan (4:1:1:2, v/v).) were then transplanted into right knee defect in each study group (five rats/group). Left knees were transplanted with corresponding hydrogels without hUCB-MSCs as controls. At 16 weeks post-transplantation, degrees of cartilage repair were evaluated macroscopically and histologically using Masson’s Trichrome, safranin-O, Sirius red staining, and type-II collagen immunostaining.
Overall, group A with 4% hyaluronic acid hydrogel resulted in superior cartilage repair grossly and histologically and achieved a cellular arrangement and collagen organization pattern mimicking adjacent uninjured articular cartilage. Immunostaining and safranin-O staining also revealed that group A displayed the largest areas of type II collagen staining. Sirius red staining revealed that the organization pattern of collagen bundles was more similar to normal cartilage in group A. No evidence of rejection was found.
The results of this study suggest that hUCB-MSCs could be used to repair articular cartilage defects in vivo and that hyaluronic acid is an attractive hydrogel candidate for use in combination with hUCB-MSCs.
KeywordsArticular Cartilage Hyaluronic Acid Pluronic Cartilage Repair Chondrogenic Differentiation
autologous chondrocyte implantation
human umbilical cord blood
human umbilical cord blood derived mesenchymal stem cells
lower critical solution temperature
modified O’Driscoll score
mesenchymal stem cell
Progress in cell biology and biomaterial technology has led to the therapeutic application of tissue engineering for the repair of cartilage defects. Mesenchymal stem cells (MSCs) have been well established as a potent cell source in the tissue regeneration field. In terms of chondrogenesis, MSCs originating from bone marrow (BM) and adipose tissue have been shown to require a biological environment stimulated by growth factors, which allows them to differentiate into hyaline cartilage tissues. Many previous studies have reported that MSCs originating from different tissue sources, such as BM or adipose tissue, can differentiate into chondrocytes under certain culture conditions when stimulated by various growth factors [1, 2, 3, 4].
Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have emerged as an alternative cellular source because they offer several advantages, such as non-invasive collection, hypo-immunogenicity, superior tropism and differentiation potential [5, 6]. By virtue of these properties, pre-clinical trials with hUCB-MSCs have been conducted in the contexts of Alzheimer’s disease , myocardial infarction , stroke  and broncho- pulmonary dysplasia (BPD) . However, the effects of hUCB-MSCs on cartilage repair have not been fully evaluated.
In terms of material requirements in regenerative medicine, hydrogels have long received attention because they serve as scaffolds that provide structural integrity and bulk for cellular organization and morphogenic guidance, function as tissue barriers and bioadhesives, can deliver bioactive agents that encourage natural reparative processes, and can encapsulate and deliver cells . With regard to cartilage tissue engineering, hydrogels have several advantages, such as high cell seeding efficacies, the abilities to transport nutrients and products to cells, the facility for straightforward modification with cell adhesive ligands and injectability in vivo as a liquid that gels at body temperature and rebuilds the three-dimensional structure [12, 13].
For proper hydrogel selection in regenerative medicine, several factors need to be considered. Among them, the ability to mimick the natural cellular environment as well as clinical availability are considered the most important factors. Hydrogels need to be physically stable in situ, allow appropriate neovascularization and vascular remodeling, and be biodegradable to enable gradual cellular ingrowth. Furthermore, they should be biocompatible and provide an appropriate three-dimensional environment for tissue growth and cellular proliferation to promote tissue regeneration .
Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan (GAG) that is present in all mammals and contains repeating disaccharide units composed of (β-1,4)-linked D-glucuronic acid and (β-1,3)-linked N-acetyl-D-glucurosamine. HA is particularly suitable for tissue engineering applications because of its high viscoelasticity and its space filling properties, and has already been utilized for the treatment of osteoarthritis . For application in cartilage repair, HA hydrogels have been shown not only to support and maintain chondrocyte viability and phenotype when cultured in vitro and in vivo but also to support and promote the chondrogenic differentiation of MSCs [15, 16, 17, 18]. Since the ideal hydrogel design should support the distribution of extracellular matrix by diffusion as well as maintain certain mechanical properties, preliminary studies were performed on various concentrations of HA. Pluronic, poly (ethylene oxide)-b-poly (propylene oxide)-b-poly (ethylene oxide) is a thermosensitive synthetic polymer, which can form gels above its lower critical solution temperature (LCST) due to hydrophobic interactions between its poly (propylene oxide) moieties. Chitosan is composed of relatively simple glucosamine and N-acetyl glucosamine units, and is a well known biocompatible material with a chemical structure similar to those of diverse GAGs in articular cartilage .
Several studies have described the effect of hydrogel systems for promoting cell proliferation, differentiation and gene expression for articular cartilage regeneration [19, 20, 21]. However, most of these studies were performed in vitro, and, to the best of our knowledge, no study has yet evaluated the feasibility and efficacy of hydrogels in vivo for cartilage repair using hUCB-MSCs. Since the cell delivery carrier is a key factor in the success of stem cell based cartilage regeneration and each different hydrogel has its own specific advantages and disadvantages, it is essential that the potentials of different hydrogels are investigated. Therefore, the purpose of this study was to explore the feasibility of cartilage repair using composites of hUCB-MSCs and four different hydrogels in a rat model.
Isolation of hUCB-MSCs
Human umbilical cord blood (hUCB) was collected from umbilical veins after neonatal delivery by an independent cord blood bank with informed consent from pregnant mothers. The hUCB-MSCs were isolated and characterized at the cord blood bank according to a previously described method  and donated for this animal study. Mononuclear cells were isolated by density gradient centrifugation at 550 × g for 30 minutes in Ficoll Hypaque (density 1.077 g/ml, Sigma, St. Louis, MO, USA). Separated mononuclear cells were then plated in α-minimum essential medium (α-MEM, Gibco BRL, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS, HyClone, Logan, UT, USA), and maintained at 37°C in a humidified 5% CO2 atmosphere with culture medium changes twice weekly. About two weeks after plating, fibroblast-like adherent cells were observed. When monolayers of MSC colonies reached 80% confluence, cells were trypsinized (0.25% trypsin, HyClone), washed and resuspended in culture medium (α-MEM supplemented with 10% FBS).
Twenty healthy, 18-week-old, male Sprague–Dawley rats were used in this study. Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of our institution. This study also followed the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Gross and histological evaluations
After sacrifice, each rat was placed on an operating table, shaved around the knee joint area, and arthrotomy was performed in the same manner as during transplantation to re-inspect the intraarticular structure. After investigation of the possibilities of rejection or infection, such as severe inflammation or extensive fibrosis, or any other abnormality, the degree of cartilage repair was grossly assessed. Coloration, luster, irregularity, presence of any depression or bulging of repaired tissues in the defect area and state of the border with adjacent normal cartilage tissue were carefully examined and gross appearance was scored following a modified Carranze-Bencano scoring system .
For histological analysis, each specimen was stained with Masson's Trichrome (Sigma, St. Louis, MO, USA), safranin-O (Sigma), Sirius red (Sigma), and immunohistochemically stained for Type-II collagen (Millopore Corp., Billerica, MA, USA), according to the manufacturer’s instructions. Full-thickness samples (cartilage and bone) were taken from each group at 16 weeks post-implantation. Samples were fixed in 10% formaldehyde, decalcified in 10% nitric acid for three days, dehydrated in graded ethanol, and embedded in paraffin wax. Paraffin-embedded sections (4 μm) were then cut and deparaffinized.
For Masson’s trichrome staining, tissue sections were stained in Masson’s composition solution for five minutes and differentiated in 5% phosphotungstic acid for ten minutes. Tissue sections were then stained in aniline blue solution for fve minutes, and excess stain was removed by washing with 0.2% acetic acid. For safranin-O staining, sections were stained with a 0.1% safranin-O solution (Sigma).
Type II collagen was detected by immunostaining. Sections were de-paraffinized, washed with PBS and treated with 0.3% (v/v) hydrogen peroxide in methanol for 15 minutes to inactivate the endogenous peroxidases. Sections were incubated with anti type-II collagen monoclonal antibody (1:200; Millipore Corp., Billerica, MA, USA) at 4°C overnight. After washing in PBS, the sections were incubated for one hour with horseradish peroxidase (HRP) anti-mouse secondary antibody. The reactivity was detected by using the EnVision TM FLEX System-HRP (DAKO, Carpinteria, CA, USA) and developed using 3,3′-diaminobenzidine chromogen (DAB, Vector Laboratory, Burlingame, CA, USA) substrate kit according to the manufacturer’s instructions.
For Sirius red staining, slides were baked at 55°C for one hour and then hydrated using xylene and graded ethanols (100%, 95%, 85%, 75%, 60% and 50%) to distilled water. Slides were then stained for two hours in saturated picric acid containing 0.1% Sirius red F3BA (Sigma). After staining, slides were removed, washed in 0.01 N hydrochloric acid for two minutes, rapidly dehydrated using a graded alcohol series (starting at 70%) to xylene, and finally cover slipped in Permount (ThermoFisher Scientific, Waltham, MA, USA). Samples stained with Sirius red staining were examined under a polarized microscope to observe the organization patterns of collagen fibers.
Sections were analyzed semi-quantitatively using the O’Driscoll scoring system . The nature of the predominant tissue (cellular morphology and safranin O staining of the matrix) was assessed by Masson’s trichrome and safranin O stainings. Structural characteristics (surface regularity, structural integrity, thickness, bonding to the adjacent cartilage), freedom from cellular changes of degeneration (hypocellularity, chondrocyte clustering) and freedom from degenerative changes in adjacent cartilage were analyzed using Masson’s trichrome stained images. All samples were scored independently by two observers and the scores were denoted by percentages. Statistical analysis was performed using the Mann–Whitney U test (SPSS, version 14) and P <0.05 was considered significant.
At 16 weeks post-transplantation, no abnormal findings suggested rejection or infection, such as severe inflammation or extensive fibrosis in 18 rats. In groups A and B experimental knees with cells, neo-formed tissues were translucent, and a smooth and intact surface with less irregularity was observed. However, depression of repaired tissues in the defect area was often present in control knees without cells. In groups C and D, the articular surfaces of the defect site in experimental knees were relatively smoother than in control knees, and had coloration closer to that of surrounding normal cartilage than to that of control knees. Furthermore, border areas of defects were less distinct and depressions were less obvious in experimental knees than in control knees (Figure 1B). Gross appearance scoring is shown in Figure 1C and experimental knees had higher scores compared to control knees in all of the four groups (* P <0.05). No significant macroscopic differences were observed among the four different hydrogel groups.
In the current study, we tried to evaluate the role of hUCB-MSCs in cartilage repair as a novel cell source. In addition, different hydrogels were evaluated to decide the most suitable one in cartilage repair. hUCB-MSC were isolated, mixed with four different hydrogels and implanted onto rat cartilage defect sites. After 16 weeks, group A with hUCB-MSCs and 4% HA showed superior cartilage repair histologically and morphologically.
Articular cartilage is highly differentiated, avascular tissue with a low self-regeneration capacity. Many researchers have attempted to increase the repair potential of damaged cartilage using cell-based therapies, such as autologous chondrocyte implantation (ACI) [25, 26, 27] or stem cell implantation [1, 6, 28, 29, 30, 31, 32]. ACI did not present the risk of immune rejection and showed significantly better histological scoring compared to the control knee [26, 27]. With MACI (matrix-induced ACI), cartilage repair improvement was also reported . However, their use is limited by the invasiveness of cell harvest requiring two operations, cell senescence from the long-term culture and difficulties associated with cell expansion. The biological activities of autologous cell cultures are dependent on patient age and genetic background, which may result in variable therapeutic outcomes. Therefore, allogenic applications including hUCB-MSCs offer an alternative option in cell therapy, because of their availability from cord blood banks, the non-invasive nature of collection, their suitability for immediate transplantation and their hypo-immunogenic properties [5, 34]. Cartilage repair has been achieved with BM and adipose derived stem cell (ADSC) implantation [1, 28, 29, 30, 31, 32, 35, 36]. However, bone formation was often observed with BM-MSCs [31, 35] and decreased cartilage repairing potency was reported with ADSC-MSCs [37, 38]. The present study shows no osseous transformation and no evidence of degeneration of the repaired cartilage up to 16 weeks. This could be a possible advantage of hUCB-MSCs. Although several studies have demonstrated the chondrogenic differentiation potential of hUCB-MSCs at the in vitro level [39, 40, 41], we could not find a report in the literature with reasonable evidence of cartilage regeneration using hUCB-MSCs in vivo. The results of this study suggest that hUCB-MSCs could be used for repair of articular cartilage defects in vivo.
In explanation of the cartilage repair mechanism with hUCB-MSCs, we could not conclude whether hUCB-MSCs regenerated the cartilaginous tissue directly or played a supportive role. Regarding the application of stem cells in regenerative medicine, several research groups have focused on the differentiation potential of stem cells, and many previous studies have demonstrated the chondrogenic potential of MSCs involved in cartilage regeneration [2, 42, 43, 44]. Likewise, the repair tissue in the present study could have been produced from the chondrogenic differentiation of hUCB-MSCs. In addition to their differentiation potential, the paracrine action of MSCs is now believed to play a role in their therapeutic effects. For example, the secretion of growth factors and cytokines has been studied [3, 45, 46]. During the repair process, unidentified factors secreted from hUCB-MSCs may have stimulated chondrogenic differentiation or enhanced cartilage-specific extracellular matrix (ECM) synthesis by bone marrow stem cells. Yang et al. demonstrated possible interactions mediated by secreted proteins between human nucleus pulposus (NP) cells and MSCs . Alternatively, the stimulus from MSCs in the host could modulate the therapeutic activities of transplanted MSCs. For example, secreted proteins from mature human articular chondrocytes suppressed MSC hypertrophy during chondrogenesis [4, 48, 49], which suggests that an interaction between hUCB-MSCs and host cells plays an essential role in cartilage regeneration. However, the key secreted factors have yet to be identified.
In addition, the anti-inflammatory and immune-modulating properties of hUCB-MSCs may also be involved in the cartilage repair mechanisms [50, 51, 52]. Singer et al. and Wang et al. demonstrated the immune modulatory function of MSCs, especially their anti-inflammatory effect, which may have affected the intraarticular microenvironment in this study and provided a suitable condition in which the transplanted hUCB-MSCs produced more relevant repair tissue even in a xeno graft trial with immunocompetent animals as in the present study [52, 53]. Taken together, we believe that the mechanism of cartilage regeneration in our model is associated with chondrogenic differentiation, the paracrine action of hUCB-MSCs, and their immunomodulatory effects.
The cell concentration for the regeneration of the defect may also be an issue. In our previous study to determine the optimal cell concentration (in preparation for submission to a journal), we transplanted 0.1, 1.0 and 10 × 107 cells/ml for cartilage repair in vivo and 1.0 × 107 cells/ml showed the best result. Based upon this result, we transplanted 1.0 × 107 cells/ml. Erickson et al.  encapsulated 60 × 106 cells/ml as well as 20 × 106 cells/ml to the methacrylated hyaluronic gels and revealed the correlation of the cell numbers with chondrogenesis and mechanical results. However, they used bovine BM-derived mesenchymal stem cells which are completely different from hUCB-MSCs. Therefore, direct comparison of these two studies in terms of the cell numbers seems to be somewhat difficult.
In the present in vivo study, we tried to identify a suitable and effective vehicle of cell delivery for cartilage repair. Our results indicated that cartilaginous tissue repairs were better overall in group A (the HA group) than in groups B, C or D. The superiority of the HA-based hydrogel correlates with a previous finding that HA has a beneficial effect on cartilage regeneration in vivo. In specific, HA has biological cues to promote chondrogenesis; therefore, local progenitor cells might migrate to the HA as well. Pluronic hydrogel contains a unique thermosensitive property among the clinically available material. Therefore, we tried to evaluate pluronic hydrogel in cartilage repair. Usually a high concentration (20% to 25% w/v) of pluronic is required for in situ gelation, and a combination of pluronic with other in situ gelling- or viscosity-enhancing polymers may enable reduction in the concentration of individual polymers and also side effects without compromising the in vitro gelling capacity . Therefore, we used 3% alginate:30% pluronic (1:1, v/v) as an in situ gelling polymer in group B, and 3% alginate:20% pluronic (1:1, v/v) in groups C and D combining with other polymers. Haider et al., showed good cell viability at this range of alginate pluronic hydrogel . A few previous studies have compared different types of hydrogels to assess the influence of scaffold chemistry on chondrogenesis [15, 57, 58]. Chung et al. compared HA hydrogels with the relatively inert poly(ethylene glycol) (PEG) hydrogels and observed a 43-fold up-regulation of type II collagen of MSCs in HA over PEG in an in vivo culture study . Wang et al. also examined the influence of four different hydrogels on ECM synthesis over a two-week period and the results showed that pluronic-collagen accumulated very little GAG or collagen, which seems to be in accordance with the results of the present study .
In conclusion, the results of this study suggest that hUCB-MSCs could be used for repairing articular cartilage defects in vivo, and HA is an attractive hydrogel candidate for hUCB-MSCs based cartilage tissue engineering.
This research was supported by the Ministry of Health and Welfare (#HI13C0451). The funding sources had no involvement in the study design, collection, analysis or interpretation of the data, writing of the manuscript or in the decision to submit the manuscript for publication.
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