Chondrocytes behaviors within type I collagen microspheres and bulk hydrogels: an in vitro study

Abstract

Cell niche, which is considered to be critical to the proliferation and differentiation of cells, is one of the most important aspects for the design and development of ideal scaffolds in tissue engineering. The mass transfer property of the scaffold affects the nutrient supply and exchange of the other substances. In this study, we prepared collagen hydrogels in the form of microspheres (CHMs) and bulk (CHB) to investigate the mass exchange differences and their influence on embedded chondrocytes. CHMs were developed by the emulsion method, which was efficient to load cells. Bovine serum albumin (BSA) was used as a diffusion model in the CHMs and CHB to evaluate the transport property of the hydrogels and the release kinetics. During the 4 week in vitro culture process, the contraction of the hydrogels, the cell viability and morphology, and the DNA and glycosaminoglycan (GAG) content were monitored at different intervals. The results suggested that the CHMs showed an obvious superiority in the transfer property over the CHB, leading to better maintenance of the chondrocyte phenotype in CHMs at the early stage of the in vitro culture. Histological analyses indicated that lots of lacunae and homogeneous positive GAG staining appeared in the CHMs from day 7. In contrast, only a few lacunae and obscure GAG staining were found in the outer area of the CHB after day 21. Without enough nutrients, the chondrocytes in the inner area of the CHB had little secreted matrix. Based on the presented CHM system, a further developed construct is suggested as a promising alternative toward the clinical application of engineered cartilaginous tissue.

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1. Introduction

Once damaged, cartilage can hardly be repaired spontaneously due to the lack of cells and nutrition.¹ In recent years, autograft and microinvasive techniques have become major available treatments, but the final effect is not greatly satisfying.² Tissue engineering is considered to be one of the most promising treatments for cartilage defect repair and regeneration.³ It has been proved that chondrocytes can secrete cartilaginous matrix in hydrogel scaffolds.⁴ Hydrogels not only supply the implanted cells with a three-dimensional microenvironment, but also act as an artificial extracellular matrix (ECM) to maintain the phenotype and function of cells.^5,6

Collagen is the main component of the ECM in cartilage. It is regarded as a superior natural polymer for tissue engineering because of its excellent properties, such as biodegradability and biocompatibility.^4,7,8 Although type II collagen is mainly contained in cartilage, there are no significant differences between the biological effects of collagen type I and II observed on the chondrogenesis of chondrocytes.⁹ Chondrocytes have a high proliferation rate in collagen hydrogels, but they might tend to lose their phenotype after long term culture.¹⁰ These dedifferentiated chondrocytes cannot synthesize the specialized extracellular matrix that is significant for cartilage defect repair and regeneration. Therefore, it is important to maintain the phenotype of chondrocytes by supplying a suitable microenvironment, including the cell–cell and cell–matrix interactions.^11,12

The limited mass exchange of large bulk materials might be a main reason causing the incomplete differentiation or dysfunction of chondrocytes due to insufficient nutrients and accumulated metabolites, especially when the cell density and crosslinking density in the scaffold are high.^13,14 Small size scaffolds, such as microspheres, can overcome these obstacles to some extent. Meanwhile, the shapes and sizes of defect areas on cartilage are usually irregular, so it may be wise to fit the defects by accumulating micron-sized materials rather than the traditional bulk ones.^4,15 As reported in the literature, microspheres are generally made from synthetic polymers and/or natural polymers in the form of hydrogels or sponge scaffolds, and are mainly applied in drug delivery or serve as cell carriers.^16,17 Cells can be seeded into hydrogels during fabrication, while it is impossible for sponges to have cells planted into them during the preparation process. A major difference between those two forms of materials is the different growing status of cells, which will affect the cellular behavior. Hydrogels offer three-dimensional (3D) microenvironments, but sponges are based on two dimensions. In the 3D system, chondrocytes will keep the natural spherical morphologies.¹⁸ Hydrogel microspheres can be fabricated by three main methods: microfluidics, droplets on non-adhesive surfaces and the emulsion method. Hong et al. developed a microfluidic material-processing chip to produce collagen microspheres.¹⁹ Hui et al. made microspheres with 3–100 μL collagen solutions of different concentrations (0.5, 1, 2 or 3 mg mL⁻¹) by pipetting droplets into a Petri dish.²⁰ But the productivity of these methods is limited, especially when the viscosity of the materials is high.^13,14

In this study, we prepared collagen hydrogel microspheres (CHMs) with chondrocytes implanted by a water-in-oil emulsion method. The preparation process was designed to be mild, and the parameters of this method were optimized. Collagen hydrogel bulk (CHB) was prepared to serve as a control. The transfer properties of the CHMs and CHB were carefully investigated and the influence on implanted cells was compared.

2. Experimental methods

2.1. Chondrocyte isolation and culture

Chondrocytes were isolated from new-born New Zealand white rabbits. Cartilages were collected and treated with 0.25 mg mL⁻¹ of trypsin for 30 minutes and 2 mg mL⁻¹ of collagenase type II for 6 hours at 37 °C. After being filtered through a 100 μm strainer and centrifuged, chondrocytes were re-suspended in alpha-modified Eagle’s medium (α-MEM, Hyclone, Beijing) containing 10% of fetal bovine serum (FBS, Gibco, USA), 1% of vitamin C and antibiotics (penicillin 100 μ mL⁻¹, streptomycin 100 μ mL⁻¹) and cultured in 5% CO₂ at 37 °C. All protocols involving animals were approved by the institutional ethical committee.

2.2. Fabrication of the CHMs and CHB

Collagen type I was extracted from calf skin with pepsin in acetic acid. Collagen hydrogel microspheres (CHMs) were made using a water-in-oil emulsion method. Briefly, the collagen solution was neutralized by 1 M NaOH in an ice bath and had a final concentration of 6.5 mg mL⁻¹. The neutralized collagen solution was injected into precooled polydimethylsiloxane (PDMS) and stirred at 500 rpm by a magnetic stirrer in an ice bath. After 10 min of emulsification, the system was moved into a water bath at 37 °C and kept under stirring for 30 min. The CHMs were produced during this stirring process. Then, the CHMs were collected by gentle centrifugation and washed three times with PBS . To prepare the cell-loaded CHMs, cells were evenly suspended in a neutralized collagen solution and then the same method as described above was followed. To investigate the effect of the cell density on the size of the CHMs and cell-loading efficiency, the final cell densities were set to 1.0 × 10⁶ cells per mL, 5.0 × 10⁶ cells per mL and 1.0 × 10⁷ cells per mL. Collagen hydrogel bulk (CHB) and cell-loaded CHB were prepared by gelation of a 100 μL neutralized collagen solution in a cylindrical mold (diameter 6.4 mm, depth 3.1 mm) with a concentration of 6.5 mg mL⁻¹ at 37 °C for 30 min. Only CHMs and CHB with a cell density of 5.0 × 10⁶ cells per mL were chosen for a 4 week in vitro culture.

2.3. Size and size distribution of CHMs

The collected CHMs were distributed in PBS buffer and imaged under a light microscope. Images were taken at random areas (more than 10) in the dish and the sizes were measured with a software (NanoMeasurer 1.2). Accordingly, the size distribution was calculated by OriginPro 9.0 (OriginLab Corp.).

2.4. Morphology of the hydrogels

The hydrogels were washed with PBS and fixed with 0.25% glutaraldehyde at 4 °C overnight. Then the hydrogels were dehydrated in a series of graded ethanol solutions and dried in a critical point dryer after using isoamyl acetate to replace ethanol. The morphology of the hydrogels was observed by scanning electron microscopy (SEM, Hitachi S-4800, Tokyo, Japan) after coating with gold using ion sputtering.

2.5. Mass transfer properties of the hydrogels

To better understand the transfer properties of the CHMs and CHB, bovine serum albumin (BSA, 66.43 kDa) was used as a release model in the hydrogels. The neutralized collagen solution was mixed with the BSA solution. The concentration of collagen was 6.5 mg mL⁻¹ and the final concentration of BSA in the hydrogels was 1600 μg mL⁻¹. The BSA-loaded CHB and CHMs were prepared the same way as the cell-loaded hydrogels. Three CHB samples (300 μL) and 3.7 mL of PBS were inserted into a 10 mL Eppendorf tube. To attain the same release condition with CHB, the CHMs made from 300 μL collagen solution were suspended in PBS with a final volume of 4 mL and were pipetted into a 10 mL Eppendorf tube. All tubes were put in the incubator at 37 °C. 500 μL of the incubated solution was sampled for measurement at intervals of 10, 20, 30, 40, 50 min, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 18, 24 and 48 h. 500 μL of fresh PBS was added into the tubes to balance the volume at each interval. The BSA concentrations in the sampled 500 μL liquid were measured by the Coomassie brilliant blue method with an ultraviolet spectrophotometer. The release ratio of BSA is calculated as follows: R = C/C_max × 100%, where R is the release ratio, C is the cumulative concentration of BSA in the buffer, and C_max is the total concentration of BSA in the testing system. Based on the acquired data, the releasing kinetics were studied using the first-order exponential decay equation (ExpDec1, y = y₀ + A × exp(−x/t)).

2.6. Contraction of the hydrogels

During the in vitro culture process, the sizes of the CHMs and CHB were recorded by a digital camera under a light microscope on days 0, 1, 2, 3, 4 and 5. The hydrogel size was measured with a software (NanoMeasurer 1.2) based on the microphotographs. The diameters of the hydrogels were calculated and the contraction ratios were investigated.

2.7. Cell viability and cell morphology

The viability of cells in the hydrogels was assessed by fluorescence staining after culturing for 1, 3, 7 and 14 days in vitro. The CHM and CHB samples were obtained and washed three times with PBS for 5 min and then immersed in PBS containing fluorescein diacetate (FDA, 1 μg mL⁻¹, Topbio Science, Beijing, China) and propidium iodide (PI, 1 μg mL⁻¹, Solarbio, Beijing, China) for 10 min. The samples were washed again with PBS and imaged by confocal laser scanning microscopy (CLSM, TCS SP 5, Leica).

2.8. Biochemical analysis

To understand the proliferation of cells in the hydrogels at the early stage of the in vitro culture (days 0, 0.5, 1, 3), both CHMs and CHB were sampled to investigate the DNA content. Both the DNA and glycosaminoglycan (GAG) content were detected in the CHM and CHB samples with longer culture periods (days 7, 14, 21 and 28). All the hydrogel samples were washed three times with PBS, and then freeze-dried at −60 °C. The lyophilized samples were carefully weighed using a Mettler Toledo XP205 (with a weigh resolution of 0.01 mg) and then digested in a 0.1% papain solution at 60 °C for 12 hours. The clear supernatants were collected after centrifugation at 4000 rpm and then divided into two parts for the tests of the DNA and GAG content. The DNA content was measured using Hoechst 33258 (B1302, Sigma). Briefly, 10 μL of the supernatant was added into 2 mL of a Hoechst 33258 solution and measured by fluorometry. A Blyscan sGAG assay kit (B100, Biocolor) was used to measure the GAG content. Briefly, 20 μL of the supernatant was added into 1 mL of Blyscan dye reagent and mixed in a shaker for 30 minutes. Then, the unbound dye was removed by centrifugation. After dissolution in the dissociation reagent, the dye content was measured by using a Varioskan™ Flash Multimode Reader (Thermo Scientific, USA).

2.9. Histological analysis

After culture for 1, 4, 7, 14, 21 and 28 days in vitro, the hydrogel samples were harvested and fixed with 4% paraformaldehyde for 1 h at 4 °C. The samples were washed three times with PBS to remove paraformaldehyde and then frozen sectioned with a thickness of 20 μm. Since the inner and outer areas of the CHB show different histological conditions, the sections obtained from the inner or outer areas of the CHB were identified and marked as CHBi and CHBo, respectively. The sections of the CHMs, the CHBi and CHBo were stained by hematoxylin–eosin (HE) and toluidine blue (TB) to investigate the cell morphology and extracellular matrix (ECM).

2.10. Statistical analysis

The GAG and DNA content and diameter contraction were analyzed with a one-way ANOVA followed by Bonferroni post-hoc correction (SPSS statistics 20.0, IBM Inc.). All data are reported as mean ± standard deviation with the significance level set at p < 0.05.

3. Results and discussion

3.1. Morphology analysis

The SEM pictures (Fig. 1) show that the morphology and structure of the CHMs and CHB are equivalent. The collagen fibers in the CHMs and CHB are uniform in diameter, and some close assembled fibers can be found in the CHMs as shown in the picture. The fibers in both samples entwine to form three dimensional networks that showed similar pore sizes and porosities. Via the morphology study by SEM, the identity of the fiber sizes and network structures between the CHMs and CHB means that the collagen in the CHMs experienced no destruction during the fabrication.

Fig. 1 Morphologies of the dried CHB and CHMs, observed by SEM.

3.2. Mass transfer analysis

The release of BSA from the hydrogels reflects the diffusion situation of molecules such as nutrients, growth factors and metabolites, which are necessary for chondrocytes to proliferate and secrete matrix. Fig. 2 shows the release ratios (R) of BSA in the CHMs and CHB. The first sampling time was 10 minutes, while the R value in the CHMs and CHB was about 80.67% and 2.31%, respectively. The ExpDec1 equations fit the release process of the CHMs and CHB very well with the adj. R-squares 0.9724 and 0.9950, respectively. According to the fitting equation, the release ratio at equilibrium (R_e) of the CHMs would reach 95.90% and the times for 90% and 95% of R_e were 0.66 and 1.46 hours, respectively. In comparison, the R_e of the CHB was 68.55% and the times for 90% and 95% of R_e were 8.17 and 10.63 hours, respectively. It is obvious that the BSA in the CHB needed a longer time to migrate to the soaking solution. BSA release tests were carried out in the study of Chia et al., and a similar result was observed.²¹ They found that 90% of BSA was released from collagen microspheres within 10 min. Distinctly different results of BSA release behaviors of the CHMs and CHB reflect the different mass exchange properties of these two hydrogels. It is reasonable to consider that BSA in the central area of the CHB could not easily diffuse to the surface because of the longer distance. The different mass transfer properties between the CHMs and CHB might be one of the main reasons leading to the significantly different results in the cell culture.

Fig. 2 Release ratios of BSA and their ExpDec1 fit lines.

3.3. Influence of cell density

The mean sizes of the freshly prepared CHM samples loaded with 1 × 10⁶, 5 × 10⁶ and 1 × 10⁷ cells per mL were 222.7 ± 82.4, 202.4 ± 61.9 and 194.8 ± 66.3 μm, respectively. The size distributions and normality tests of the samples are shown in Fig. 3a. The results indicate that the average size of the CHMs slightly decreased when the cell density increased. The one way ANOVA overall analyses showed that the average sizes were significantly different at the 0.05 level. The mean comparisons between 1 × 10⁶ and 5 × 10⁶, 1 × 10⁶ and 1 × 10⁷ were significantly different, but that difference between 5 × 10⁶ and 1 × 10⁷ was not significant. Corresponding to the size analysis, the CLSM results of the freshly prepared CHM samples encapsulating cells of different densities are shown in the Fig. 3b. The pictures indicate that the CHMs can efficiently encapsulate chondrocytes at different densities, and the cells were uniformly distributed in the microspheres.

Fig. 3 The influence of cell density on the CHM size and encapsulation efficiency. (a) Size distributions of the CHMs loaded with 1 × 10⁶, 5 × 10⁶ and 1 × 10⁷ cells per mL, which were calculated based on the microsphere images under a low magnification light microscope; (b) CLSM pictures of the cell loaded CHMs. Scale bars represent 100 μm.

The CLSM pictures show that the cell viability and cell morphology had no obvious difference between the CHMs and CHB when the cells were encapsulated at the early stage of the in vitro culture. Combined with the results of SEM and CLSM together, it indicates that the emulsion method did not change the structure and biocompatibility of the collagen hydrogels or affect the viability of the cells. Based on the acquired results, it is reasonable to consider that the different cell responses in the CHM and CHB in vitro cultures were mainly caused by the different mass exchange properties of the hydrogels.

3.4. Contraction of the hydrogels

The contraction of hydrogels with cells embedded is a common phenomenon during in vitro cultures.^22–24Fig. 4 shows the diameter contraction of the cell-loaded CHMs and CHB during the initial 5 day in vitro culture. The CHMs contracted faster than the CHB. The diameters of the CHMs shrank sharply on day 1 to around 60% of the original size, and remained relatively stable afterwards. The average diameter of the CHB contracted to 85% of the original size on day 1, and continued to contract to around 45% of the original size on day 5. The contraction of the hydrogels leads to a higher cell density and a change of network structure in the hydrogels. In our study, according to the results of the contraction ratios, the cell density in the CHMs remained at a higher value than in the CHB from day 4, which could be ascribed to both the contraction and cell proliferation. The high cell density was considered to be in favor of the conservation of chondrocyte phenotypes.²⁵ Normally, chondrocytes harvested from culture dishes show a fibroblast-like morphology. After being seeded in the hydrogels, these fibroblast-like chondrocytes cause a fast contraction of the hydrogels.²⁶ The change of the morphology to round-shape not only implied the recovery of the cell function but was also the endpoint of the contraction. Meanwhile, contractions could also enhance the density of the collagen fibers and gel stiffness,¹² which could further decrease the contraction. The results in Fig. 4 indicate that the contraction of the CHMs terminated on day 1, suggesting an earlier recovery of the chondrocytes than with the CHB.

Fig. 4 Diameter contraction of the CHMs (n = 129–238) and the CHB (n = 10–11) over 5 days of in vitro culture (*p < 0.05, and **p < 0.01, same below).

3.5. Cell viability and cell morphology

If the scaffolds could balance the permeability and mechanical properties, provide good protection, dispense enough nutrients and allow a quick removal of metabolites, the suitable micro-environment for chondrocytes might result in a good phenotype recovery and high matrix production.^27,28 The cell condition was evaluated from the DNA content and CLSM pictures which were taken at different intervals. Fig. 5a shows the DNA content in the CHMs and CHB on days 0, 0.5, 1 and 3, which reflects the proliferation of the loaded cells at the early stage of the in vitro culture. After about 12 hours, the chondrocytes proliferated so exuberantly in the CHMs that the DNA content doubled and the percentage of DNA to the dry weight of the CHMs was about 2.5 times that of the CHB. The proportion (DNA/dry weight) of the CHB was unchanged until day 3, whereas that of the CHMs kept on growing, and was 1.6-fold and 0.9-fold higher than that of the CHB on day 1 and day 3, respectively. Also, the proliferation of chondrocytes in both the CHMs and CHB indicates the good cytocompatibility of the scaffold. With the increase of culture time, more GAG would be secreted in the hydrogels, leading to the inaccuracy of the proportion (DNA/dry weight). Therefore, only the first three days of the culture period were monitored.

Fig. 5 The cell viability and cell morphology in the CHMs and CHB. (a) The chondrocyte proliferation detected by DNA assay (n = 3). (b) Confocal laser scanning microscopy pictures of the CHB and CHMs loaded with 5 × 10⁶ cells per mL. Scale bars represent 100 μm.

For different culture periods, the cell morphology in the CHMs and CHB was observed using CLSM and the images are shown in Fig. 5b. According to the images, chondrocytes regained the chondrogenic phenotype and lots of cell clusters appeared in the CHMs. On day 1, most of the cells in both hydrogels kept the spherical morphology, while a small amount of them tended to show a fibroblast morphology. With the elongation of the culture time, until day 7, there was almost no spreading out of the chondrocytes in the CHMs. On the contrary, the chondrocytes in the CHB spread out and became fibriform from day 3 onwards and the apparent cell density was lower than that in the microspheres.

3.6. Biochemical analysis

The GAG secreted by the chondrocytes during the in vitro culture was abundant, especially when the cells were cultured for longer than 7 days. Fig. 6 shows the GAG content and the proportion of GAG to DNA in the hydrogels when cultured for 7, 14, 21 and 28 days. According to Fig. 6a, the GAG content in the CHMs increased significantly on day 14 with respect to that on day 7, and then gradually increased in the next 2 weeks. Meanwhile, the GAG content in the CHB showed a slow increase in the first 2 weeks, but rose up to around 7.6 times on day 21 compared to that on day 14, and then the GAG content doubled in the 4^th week. By comparing the GAG content in the CHMs and CHB, it is obvious that the content in the CHMs is always higher than that in the CHB. Especially, after being cultured for 14 days, the GAG content in the CHMs is 10-fold higher than that in the CHB (p = 0.039). The difference in GAG content between these hydrogels becomes less pronounced when the culture period extends to 28 days.

Fig. 6 Biochemical analyses of secreted GAG in the hydrogels. (a) The secreted GAG increasing in the CHMs and CHB; (b) the proportion of GAG to DNA in the CHMs and CHB (day 7, 14 n = 2; day 21, 28 n = 3).

As a major component of the extracellular matrix, the quantity of GAG secreted by chondrocytes can reflect the function expression of the cells. According to Fig. 6b, it was found that the proportions of GAG to DNA in both the CHMs and CHB increased with the culture period. The chondrocytes in the CHMs maintained the proportion of GAG to DNA on day 28. Different from the situation in the CHMs, the chondrocytes in the CHB secreted little GAG in the first 2 weeks. In comparison, the GAG/DNA values in the CHMs were significantly higher than those in the CHB, from 6.9-fold, to 2.1-fold and 1.5-fold on day 14, 21 and 28, respectively. The quantitative and qualitative analyses of GAG indicated that the secretion of GAG in the CHMs is stronger and 1–2 weeks earlier than that in the CHB.

3.7. Histological analysis

Fig. 7 shows the HE staining results of the CHMs and CHB. In the CHB, the encapsulated cells spread on day 1 and reached the highest cell density on day 4, followed by a continuous decrease in the cell density from day 7 to day 28. Furthermore, the cells maintained their shape and separated distribution, which means the phenotype was not recovered during the process. Only a few lacunae could be found in the CHB samples from day 7 to day 28. On the contrary, it can be seen that the cell density in the CHMs increased and the phenotype of the cells was spherical. A large amount of cartilage lacunae was found in the CHM samples on day 7. Then the lacunae became more obvious on day 14 and afterwards. Cells in the CHB maintained the fibroblast-like morphology with few clusters, which matches the studies made by Van Susante and Schuman.^10,11 Several clusters can be observed in the CHBo while the situation in the CHBi is even worse. This might be explained by the mass exchange difference as mentioned in the BSA release test. Nutrients could only diffuse into the outer area of the gel and were consumed by the cells in the outer area, leading to the insufficient nutrient supply and dysfunction of the chondrocytes in the inner area of the hydrogels.

Fig. 7 Histological observations of the CHB and CHMs after the in vitro culture, and staining by haematoxylin and eosin (HE). Scale bars represent 200 μm.

TB staining of the CHMs and CHB are shown in Fig. 8. On day 1 and day 4, the TB staining in both the inner area of the CHB (CHBi) and outer area of the CHB (CHBo), as well as the CHMs is negative. The first positive staining in the CHMs was observed on day 7. The color kept on becoming deeper and more homogeneous with the increase in culture time from day 7 to day 28, which means an increase in the GAG content in the CHMs. Positive staining was also found in the CHBo on day 7, but the color is light and inhomogeneous. In contrast, the CHBi showed almost no positive staining even on day 28. Only a few areas around the lacunae were positively stained. Homogeneous positive TB staining in the CHM also reflects the phenotype recovery of the chondrocytes. It was reported that matrices secreted by chondrocytes also own the ability to promote the recovery of the phenotype,¹¹ and that chondrocytes need the matrix support to retain the new matrix.²⁹ According to the results of TB staining and biochemical analyses in our study, we can infer that the chondrocytes in the CHMs have a better function expression than those in the CHB.

Fig. 8 Histological observations of the inner area of the CHB (CHBi), outer area of the CHB (CHBo) and the CHMs after the in vitro culture, and staining by toluidine blue (TB). Scale bars represent 200 μm.

The specific extracellular matrix secreted by chondrocytes plays a crucial role in cartilage regeneration and neo-cartilage formation.³⁰ It is important to supply a nice microenvironment for chondrocytes to keep the phenotype, since the dedifferentiated cells are apt at losing the capability to secrete acceptable ECM to repair the defect.^31–33 The inadequate or inappropriate secreted ECM cannot enhance the mechanical properties of engineered cartilage tissue.^8,15,34 Farrell compared the mechanical properties of different areas of the hydrogel (with a size of 4 mm in diameter, 2.25 mm in depth) and found a 2-fold decrease in the compressive strain from the outer area to inner after 21 days.³⁵ The mass transfer property, which will affect the nutrient supply and metabolic product diffusion, might be the reason that gives rise to the difference of the proliferation and morphology of chondrocytes,^2,18,36 and further leads to the mechanical differences of the engineered cartilage. Lots of studies have been made on chondrocyte–collagen hydrogel mixtures in the last decade, but most of these were bulk ones.^4,5,27 There were a few studies focusing on the permeability of hydrogels, with even less investigation executed on the relationship between mass transport and cell response in in vitro cultures. Beside the structure factors, the hydrogel size is one of the most obvious factors which has a great influence on the solute transport. Based on the study of the microspheres in this paper, we were trying to reveal the size effect of the hydrogels and guide the future research and application of hydrogels for tissue regeneration.

We got a cartilage-like tissue with a homogeneous specific extracellular matrix as shown in the TB staining results. In the study of Valentin Dhote, they mentioned that the diffusion of the matrix secreted by chondrocytes will make the matrix more homogeneous, which is the key factor in cartilage regeneration.³⁷ The results of our studies discovered a similar phenomenon. Therefore, the collagen microspheres of this study promoted the proliferation of cells at an early stage, and accelerated the function expression at the late stage of the in vitro culture. The superior permeability might be a positive factor, which could accelerate cartilage regeneration.

4. Conclusions

CHMs loaded with chondrocytes were fabricated using an emulsion method under mild conditions, and they were further compared with the CHB via a 4 week culture in vitro. With the help of the outstanding mass transfer property of the CHMs, the chondrocytes in the CHMs could not only proliferate more quickly, but also regain a normal morphology and phenotype faster than those in the CHB. As a consequence, a more specific matrix is secreted and homogeneously distributed in the CHMs. Lots of lacunae are observed when the chondrocyte-loaded CHMs are cultured for 4 weeks. In contrast, a limited matrix is accumulated in the outer area of the CHB and less in the inner area. Therefore, the size would obviously influence the mass exchange of the hydrogels and further affect the behavior of the embedded cells. The development of cartilaginous constructs based on CHMs for cartilage regeneration is under investigation both in vitro and in vivo.

Acknowledgements

This study was financially supported by the National Key Technology Research and Development Program (2012BAI42G00), the National Science Foundation of China (51403134, 31130021) and the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, China.

Notes and references

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