Chondrogenic Differentiation Could Be Induced by Autologous Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Matrix Scaffolds Without Exogenous Growth Factor.

Tissue Engineering Part A Chondrogenic differentiation could be induced by autologous bone marrow mesenchymal stem cell–derived extracellular matrix scaffolds without exogenous growth factor (doi: 10.1089/ten.TEA.2014.0491) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 1 of 34

1 Chondrogenic differentiation could be induced by autologous bone marrow mesenchymal stem cell–derived extracellular matrix scaffolds without exogenous growth factor Cheng Tang,1,2,3,﹡ Chengzhe Jin,1,2,3,﹡ Yan Xu,1,2,3 Bo Wei,1,2 Liming Wang,1,2,3

1

Department of Orthopaedic Surgery, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China

2

Cartilage Regeneration Center, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China

3

China-Korea United Cell Therapy Center, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China ﹡

These two authors contributed equally to the study

Address correspondence to: Liming Wang Department of Orthopaedic Surgery, Nanjing First Hospital Nanjing Medical University Nanjing 210006, China E-mail: [email protected] (Liming Wang)


Page 2 of 34

2 Abstract: We previously found that the combination of an autologous bone mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM) scaffold with bone marrow stimulation could enhance hyaline cartilage regeneration. We suspected that chondrogenic differentiation could be induced by the aBMSC-dECM scaffold. This study aimed to investigate whether aBMSC-dECM scaffolds could promote chondrogenic differentiation without exogenous growth factors. BMSCs were seeded on aBMSC-dECM scaffolds and cultured in vitro with or without TGF-β3 (E+ or Egroup). Atelocollagen scaffolds were used as controls (C+ or C- group). The chondrogenic differentiation was evaluated by histological, biochemical and real-time PCR assays. After 3 weeks, cartilage-like tissue with a homogeneous structure, a high cartilaginous matrix content (proteoglycan and type II collagen) and high expression levels of cartilage-associated genes (COL2A1, ACAN and SOX9) was observed in the E+, E- and C+ groups. In addition, BMSCs in each scaffold (E group or C group) were preconditioned with chondrogenic media in vitro for 1 week and then implanted in the backs of nude mice for 3 weeks. Three weeks later, cartilage matrix formation (proteoglycan and type II collagen) was achieved only in the E group, confirmed by safranin O staining and immunohistochemical staining for type II collagen. Taken together, these results indicate that aBMSC-dECM scaffolds could induce chondrogenic differentiation. Thus they could be successful candidate scaffolds for cartilage tissue engineering. Keywords:

Autologous;

aBMSC-dECM

scaffold;

BMSC;

Chondrogenic


Page 3 of 34

3 differentiation; Cartilage Introduction The autologous matrix-induced chondrogenesis (AMIC) technique is frequently used to enhance the potential for bone marrow stimulation (BMS)-based cartilage repair. In AMIC, a bioactive scaffold such as type I/III collagen scaffold or chitosan is implanted into the cartilage defect after BMS treatment.1-3 The improved long-term outcomes after using the AMIC technique have gained considerable attention.4 It has been suggested that the scaffolds could enhance the chondrogenic differentiation of endogenous bone mesenchymal stem cells (BMSCs) and guide the repair of injured tissue toward a more hyaline-like histological effect.5 However, it should be noted that the majority of the currently used scaffolds are originally derived from xenogenous tissues. In addition to raising ethical issues in clinical practice, the implantation of these scaffolds is related to an increased risk of pathogen transmission, inflammation as well as the other immunological reactions.6, 7 It is well understood that autologous scaffolds can effectively overcome these disadvantages and largely improve the quality of cartilage tissue engineering owing to their enhanced safety and efficacy.8,

9

For example, Chen et al. used poly

(D,L-lactic-co-glycolic) acid (PLGA) mesh as a template to fabricate autologous MSC-derived extracellular matrix (ECM) scaffolds and found that these promoted chondrogenic differentiation without any exogenous growth factors.8, 10 Moreover, we developed a novel autologous BMSC-derived ECM (aBMSC-dECM) scaffold without any template and found that implantation of this scaffold into osteochondral defect


Page 4 of 34

4 sites following BMS in a rabbit model enhanced hyaline cartilage regeneration.11, 12 Furthermore, previous studies have indicated that the chondrogenic potentiality of human synovium-derived stem cells could be achieved on a decellularized stem cell matrix13 and that specific cell-derived ECM scaffolds could provide various signals to control the chondrogenic differentiation of MSCs.10 However, little is known regarding whether the aBMSC-dECM scaffold itself might induce chondrogenic differentiation. In this study, in vitro culture and in vivo implantation were performed to further determine whether the aBMSC-dECM scaffold could promote chondrogenic differentiation of BMSCs without any exogenous growth factors.

Materials and Methods Isolation and Culture of BMSCs The use of laboratory animals was approved by the Institutional Animal Experiment Committee of Nanjing Medical University. The experimental protocol met the guidelines of the National Institutes of Health. In our study, five New Zealand white rabbits (aged two weeks) were euthanatized using an overdose injection of pentobarbital, then the BMSCs were isolated and cultured as reported previously.14 Briefly, the bone marrow was flushed from the tibias and femurs via phosphate-buffered saline (PBS), and mononuclear cells (MNCs) were obtained using density-gradient centrifugation with Lymphoprep (Axis-Shield, Oslo, Norway) at 800 × g for 20 min. Then the cell fractions were then resuspended with Dulbecco’s modiﬁed Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), which contains


Page 5 of 34

5 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). The isolated MNCs were then seeded (density: 1.0 × 105 cells/cm2) and cultured (37°C, in a 5% CO2 atmosphere, 95% humidity). Forty-eight hours later, the non-adherent cells were removed, and the adherent BMSCs were further cultured to grow into an aBMSC-dECM membrane. During the culture period, certain BMSCs (passage one) were collected for cell seeding. Therefore, these cells were pooled, aliquoted, and frozen in DMEM containing 20% FBS and 10% dimethyl sulfoxide.

Preparation of aBMSC-dECM Scaffolds The aBMSC-dECM scaffolds were prepared according to a previous description.12 In brief, to stimulate ECM deposition, when the primary BMSCs reached a confluence of 70–80%, 50 μg/ml L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) was then added into the culture medium. The ECM membrane was separated carefully after 4 weeks of in vitro culture and freeze-dried at −70°C under 1 Pa for 48 h on a LGJ-10D freeze-drier (Si-Huan, Beijing, China). The freeze-dried specimens were then cross-linked using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (50 mM) and N-hydroxysuccinimide (50 mM) for increasing their mechanical strength and freeze-dried again. To obtain the final form of the aBMSC-dECM scaffold, a biopsy punch and a clean razor blade were used to cut and trim into 6 mm in diameter and 2 mm in thickness.


Page 6 of 34

6 In Vitro Culture of Cell-Scaffold Constructs Shortly after the preparation of the aBMSC-dECM scaffolds, autologous BMSC aliquots were thawed and sub-cultured. The scaffolds were sterilized in 75% ethanol (10 h), washed with PBS (three times), and immersed overnight in culture media to remove all traces of ethanol. Then the BMSC suspensions (passage 3; cell density: 3 × 106/ml) were dynamically seeded on the scaffolds over 90 min with a nutator, which as reported previously.15 Atelocollagen scaffolds (Koraku, Tokyo, Japan) were used as experimental controls. After cell seeding, the cell-scaffold constructs were transferred to 6-well plates and cultured in vitro. The in vitro study comprised four groups (n = 18 each): 1) C+, BMSC-atelocollagen scaffold constructs cultured in a chondrogenic-defined medium (DMEM

supplemented

with

insulin-transferrin-selenium

mixture,

100

nM

dexamethasone, 50 µg/ml L-ascorbic acid 2-phosphate, 1.25 mg/ml bovine serum albumin, and 40 mg/ml L-proline) with 10 ng/ml TGF-β 3, a typical chondrogenic inducer;

2)

E+,

BMSC-aBMSC-dECM

scaffold

constructs

cultured

in

a

chondrogenic-defined medium with TGF-β 3; 3) C−, BMSC-atelocollagen scaffold constructs cultured in a chondrogenic-defined medium without TGF-β 3; and 4) E−, BMSC-aBMSC-dECM scaffold constructs cultured in a chondrogenic-defined medium without TGF-β 3. The constructs were collected for further analysis after 3, 10 and 21 days.

In Vivo Implantation of Cell-Scaffold Constructs


Page 7 of 34

7 For the in vivo study, the cell-scaffold constructs were prepared as above and then cultured in vitro in a chondrogenic-defined medium without TGF-β 3 for 1 week (E and C groups). Six-week-old male nude mice (n = 12 per group) were anesthetized with chloral hydrate solution (0.4 mg/g). Under sterile conditions, the backs of the mice were incised, and four constructs from the same group were implanted into the subcutaneous tissue. Four mice (carrying 4 constructs each) of each group were sacrificed to assess chondrogenic differentiation at 1, 2 and 3 weeks after implantation.16

Gross Observation and Volume Measurement The gross morphology of in vitro and in vivo constructs was assessed by shape and color. Construct firmness was tested by a pinch test. The volumes of in vitro and in vivo constructs were measured from three-dimensional images obtained by a CCD camera that was equipped with a previously described computer vision system.16, 17

Histological Analysis The in vitro and in vivo constructs were evaluated by histological staining with safranin O and von Kossa, and by immunohistochemical analysis of type II collagen expression. In brief, the constructs were fixed with 4% formaldehyde for 24 h, dehydrated and embedded in paraffin, sectioned at 4-m thickness, and then stained with safranin O and von Kossa. For von Kossa staining, the sections were incubated with 3% silver nitrate solution under ultraviolet light for 1 h, and then counterstained


Page 8 of 34

8 with toluidine blue. For immunohistochemical analysis, the sections were sequentially treated with 3% H2O2 and proteinase K, and then were incubated with a mouse monoclonal antibody against rabbit type II collagen antibody (1:100; Acris, Herford, Germany) for 1.5 h at ambient temperature. Then the sections were incubated with a biotinylated secondary antibody against mouse IgG (1:200; Maixin, Fuzhou, China) for 10 min, washed, and incubated with a peroxidase-conjugated streptavidin solution. The sections were counterstained with hematoxylin and mounted later for microscopic observation (BX53, Olympus, Japan). The percent area of positive staining was measured with a computer-assisted automated image analyzer (Image-Pro plus 6.0; Media Cybernetics Inc., Bethesda, MD, USA).18, 19 The software measured 10 random fields per slide, and calculated the ratio of the area showing positive staining to the whole area of each construct.

Real-Time Polymerase Chain Reaction (PCR) The expression level of genes associated with the cartilage matrix (COL2A1, ACAN, SOX9, COL1A2, and COL10A1) were assessed in the in vitro constructs (n = 6 each) using real-time PCR. Briefly, total RNA was extracted with TRIzol reagent (Gibco), and PCR was performed via a quantitative PCR kit (Toyobo, Osaka, Japan) and an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. All reactions were run in triplicate. The sequences of primers used in this study are as follows: COL2A1 forward 5′-CAGGCAGAGGCAGGAAACTAAC-3′,


Page 9 of 34

9 COL2A1

reverse

5′-CAGAGGTGTTTGACACGGAGTAG-3′;

ACAN

forward

5′-ATGGCTTCCACCAGTGCG-3′,

ACAN

reverse

5′-CGGATGCCGTAGGTTCTCA-3′;

SOX9

forward

5′-GTACCCGCACCTGCACAAC-3′,

SOX9

reverse

COL1A2

forward

5′-TCCGCCTCCTCCACGAAG-3′; 5′-GCGGTGGTTACGACTTTGGTT-3′,

COL1A2

reverse

5′-AGTGAGGAGGGTCTCAATCTG-3′;

COL10A1

forward

5′-ATCAGCCACTGGGAAGCC-3′,

COL10A1

reverse

5′-TTCGGTCCACTTGGTCCTC-3′;

GAPDH

forward

5′-CGTCTGCCCTATCAACTTTCG-3′,

GAPDH

reverse

5′-CGTTTCTCAGGCTCCCTCT-3′. The fluorescence intensity was recorded under the following setting: 15 s at 90°C and 60 s at 60°C, for 40 cycles. At last, the gene expression value of individual constructs was carefully calculated relative to GAPDH expression using the 2-△Ct method.19

Chemical Assays The DNA and glycosaminoglycan (GAG) contents of the in vitro constructs (n = 6 per group) were measured by chemical assay as described previously.12 Briefly, the constructs were dried (at 37°C for 48 h) and then digested (at 60°C for 24 h) with a papain solution (5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, and 125 µg/ml papain; pH 6.4), followed by centrifugation (12,000 × g, 10 min). The resultant supernatant was further used for chemical analysis of DNA and GAG contents.


Page 10 of 34

10 To determine the total DNA content, a Quit-iT dsDNA kit (Invitrogen, Eugene, OR, USA) was used. DNA from salmon testes (Sigma-Aldrich) was used to generate a standard curve. In brief, the supernatant was reacted with Hoechst dye 33258 for 30 min in the dark, following which the intensity of fluorescence was measured using a 96-well plate reader with excitation and emission wavelengths of 360 and 460 nm, respectively (Perkin-Elmer LS-55, MA, USA). GAG content was measured by a dimethylmethylene blue (DMB) colorimetric assay. Briefly, the supernatant was mixed with DMB solution for GAG binding. The GAG-dye complexes were then collected by centrifugation (12,000 × g, 10 min). The GAG content was calculated according to a standard curve of sulfate chondroitin from shark cartilage (Sigma-Aldrich) at 530 nm on a Benchmark plus microplate spectrophotometer (Bio-Rad, Tokyo, Japan).

Micro-Computed Tomography (CT) Imaging The in vivo constructs (n = 8 per group) were analyzed to assess hypertrophic changes by micro-CT equipped with a SkyScan 1072 scanner and its associated analysis software (SkyScan, Antwerp, Belgium), which was previously described.18 Briefly, the constructs were tightly enclosed to plastic wrap to minimize movement. Image acquisition was conducted at 100 kV and 98 mA, at an image resolution of 24 μm. In order to segment the constructs from the background, the same threshold was applied to the images. Two-dimensional images were used in our study to generate three-dimensional renderings. For quantitative analysis of calcified matrix volumes,


Page 11 of 34

11 each construct was manually segmented in each three-dimensional image. Then a second evaluation was performed for the calculation of the volume of the calcified matrix.

Statistical Analysis All data were expressed as mean ± standard deviation (SD). The effects of time (3, 10 and 21 days) on volume, the percent area of positive staining, and the chemical content of in vitro constructs in each group were evaluated by one-way analysis of variance (ANOVA). The differences of volume, the percent area of positive staining, and the chemical contents of the in vitro constructs among the C+, E+, C− and E− groups at 21 days, and the effects of time (1, 2 and 3 weeks) on the volume of in vivo constructs in the C and E groups were also analyzed by one-way ANOVA, followed by a post-hoc test (Student-Newman-Keuls). The volume difference of the in vivo constructs between the C and E groups at 3 weeks was compared using a Student’s t test. A P value less than 0.05 (two-sided) was considered statistically significant. All statistical analyses were carried out using SPSS 13.0 (SPSS, Chicago, IL, USA).

Results Gross Observation and Volume of the In Vitro Constructs For gross observation, the constructs were obviously different in size and shape between groups. During in vitro culture, a significant decrease in construct volume was observed in all groups except the E+ group (Fig. 1A). After 21 days, the volumes


Page 12 of 34

12 of the in vitro constructs were 1.22 ± 0.24, 10.89 ± 2.09, 0.16 ± 0.04 and 8.07 ± 0.58 mm3 in the C+, E+, C− and E− groups, respectively. The volumes of the constructs in the E− group were greater than those in the C+ and C− groups at 21 days, whereas no significant difference was found compared with those of the E+ group (Fig. 1B).

Histological and Immunohistochemical Analyses of the In Vitro Constructs Safranin O staining confirmed that cartilaginous structures were formed in the C+, E+ and E− groups. Cartilaginous structures containing lacunae and exhibiting metachromatic staining were evident in the C+, E+ and E− groups; however, strong metachromatic staining was observed in the cartilaginous structures of the E+ group (Fig. 2A). The area of positive sulfate proteoglycan staining in the C+, E+ and E− groups increased significantly over time. No significant difference was noted between the C+ and E− groups at 21 days after in vitro culture (Fig. 2B and C). The expression of type II collagen showed a similar pattern of positive staining in the C+, E+ and E− groups (Fig. 3A). The area of positive type II collagen staining increased gradually during the culture period; however, no significant difference was noted between the E+ and E− groups at 21 days (Fig. 3B and C). Cartilaginous lacuna-like structures, sulfate proteoglycan, and type II collagen staining were not observed in the C– group during the in vitro culture. The differentiated BMSCs in the E+ group showed no change during the entire period of culture. However, hypertrophic changes of BMSCs were found at 21 days in both the C+ and E− groups.


Page 13 of 34

13 Expression of Cartilage Matrix-associated Genes from the In Vitro Constructs Real-time PCR was used to evaluate mRNA expression in the in vitro constructs. The expression level of cartilaginous markers (COL2A1 and ACAN), a key chondrogenic regulator (SOX9), a key hypertrophic marker (COL10A1), and a key differentiation marker (COL1A2) were assessed in the in vitro constructs. On day 3, cells in all groups showed low expression of COL2A1, which encodes type II collagen. During in vitro culture, the E+ and E− groups showed constantly increasing expression levels of COL2A1 (Fig. 4A) and decreasing expression levels of COL1A2 (Fig. 4D), which encodes type I collagen. COL2A1 expression was higher in the C+ group than in the E− group, but was downregulated by 50% on day 21 (Fig. 4A). The expression of ACAN, which encodes the core protein aggrecan, increased over time in groups C+, E+, and E−, whereas its expression was downregulated in the C− group (Fig. 4B). SOX9 expression followed the same trends as those observed with respect to the changes in COL2A1 and ACAN expression in each group. Specifically, its expression level was upregulated over time in the E+ and E− groups, but decreased in the C+ and C− groups (Fig. 4C). The expression of COL10A1, which encodes type X collagen, was higher in the C+ group than in the E+ group at different time points, whereas its expression was the highest in the E− group on day 21. In contrast, COL10A1 maintained a very low expression level in the C− group throughout in vitro culture (Fig. 4E).

Chemical Assessments of the In Vitro Constructs The total DNA content of each construct was maintained at a high level in the E+


Page 14 of 34

14 and E−groups during the in vitro culture, but decreased over time in the C+ group (Fig. 5A). The DNA content was significantly lower in the C+ group than in both the E+ and E− groups on day 21 (both P < 0.05; Fig. 5a). The amount of GAG significantly increased with time in the C+, E+, and E− groups (Fig. 5B); however, the E+ and E− groups had a higher amount of GAG than the C+ group on day 21 (both P < 0.05; Fig. 5b). The GAG/DNA ratio increased gradually over time in all groups (Fig. 5C); however, this ratio was significantly higher in the E−group than in the C+ group on day 21 (P < 0.05; Fig. 5c). The DNA and GAG content of the constructs could not be measured in the C- group because of the small sample weight.

Gross Observation and Volume Measurement of the In Vivo Constructs The in vivo constructs (Fig. 6D and 6E) in the E group showed whitish, hyaline cartilage-like morphology after 1 and 2 weeks; however, their surfaces (Fig. 6F) hardened after 3 weeks. The in vivo constructs in the C group (Fig. 6A-C) appeared as gray fibrous-like tissue. The constructs of the E group maintained their size and shape at baseline during the in vivo implantation, whereas the volume of the constructs significantly decreased in the C group. The estimated volume of the construct was significantly larger in the E group than in the C group (6.67 ± 0.87 vs. 0.09 ± 0.03 mm3; P < 0.001) at 3 weeks after in vivo implantation (Fig. 6G).

Expression of Chondrogenic Markers of by the In Vivo Constructs The chondrogenic differentiation of the in vivo constructs was confirmed by


Page 15 of 34

15 safranin O staining and immunohistochemical analysis of type II collagen. The accumulation of sulfate proteoglycan and type II collagen was observed in the E group (Fig. 7A). No significant difference was observed in distribution or intensity between the outer and inner regions of the E group at 1 week. After 2 weeks, the expression of sulfate proteoglycan and type II collagen was decreased slightly, especially in the outer region (Fig. 7B and C). No positive sulfate proteoglycan and type II collagen staining was found in the C group during the entire implantation period. Notably, in morphology, the differentiated cells in the E group were predominantly rounded; these cells were encapsulated in lacunae, which was similar to chondrocytes in native cartilage. These round shapes and lacunar structures were not observed in the C group.

Expression of Hypertrophic Markers in the In Vivo Constructs Three-dimensional reconstruction and quantitative analysis using micro-CT indicated that calcified matrix formation was formed first in the outer regions of the constructs in the E group at 2 weeks after implantation, and then spread to the central region, corresponding with an increased volume of the calcified matrix after 2 weeks. No calcified matrix was observed in the C group (Fig. 8A and B). Von Kossa staining further confirmed that a hypertrophic change occurred in the E group. Black staining, indicative of calcified mineral deposits, was found in the outer regions of the E group and spread gradually to the central regions, with the positive area gradually increasing throughout the in vivo implantation period (Fig. 8A and C).


Page 16 of 34

16

Discussion In this study, we attempted to determine whether the aBMSC-dECM scaffold could promote chondrogenic differentiation of BMSCs without any exogenous growth factors. The in vitro constructs were observed with a homogeneous structure, high cartilaginous matrix content, and high expression level of cartilage-associated genes in both the E+ and E− groups. The in vivo constructs showed cartilage formation in terms of accumulation of cartilage matrix only in the E group. These results suggested that the aBMSC-dECM scaffold possessed chondrogenesis-inducing activity, thereby promoting chondrogenic differentiation of autologous BMSCs. Multipotent BMSCs that are able to differentiate into chondrocytes have been successfully used for cartilage tissue engineering.20 Nevertheless, a major limitation of cartilage tissue engineering with BMSCs is that exogenous growth factors are required to induce chondrogenic differentiation.12,

21

One of the most common

strategies to achieve this is the incorporation of growth factor-loaded microparticles into the tissue engineering materials. However, this does not mimic the natural presentation of the factors, and this approach has been associated with the uncontrolled release of growth factors that are not normally secreted outside of cells. Furthermore, the latter application raised regulatory issues related to binding factor conformation and activity.22-24 Bioactive scaffolds have been expected to play a crucial role in the induction of chondrogenic differentiation.25 Compared with atelocollagen scaffolds, in this study,


Page 17 of 34

17 only aBMSC-dECM scaffolds underwent chondrogenesis and accumulated large amounts of cartilaginous matrix (consisting of sulfate proteoglycan and type II collagen) when cultured without TGF-β3 in vitro. In addition, BMSCs in aBMSC-dECM scaffolds upregulated their expression of cartilaginous genes (COL2A1, ACAN, and SOX9) irrespective of whether TGF-β3 was added into the culture media. These findings were further confirmed by our in vivo study, which demonstrated that when the cell-scaffold constructs were implanted into the subcutaneous tissue of nude mice, chondrogenic differentiation occurred in the E group whereas no evidence of chondrogenesis was found in the C group. These data indicate that the aBMSC-dECM scaffold can support chondrogenic differentiation of BMSCs without the requirement of exogenous growth factors. Choi et al. reported that a porcine chondrocyte-derived ECM scaffold could not only support chondrogenic differentiation of rabbit BMSCs in vitro but also delay the degeneration of

chondrogenic

phenotypes

in

vivo21,

suggesting

that

such

a

porcine

chondrocyte-derived ECM scaffold might provide a favorable and native cartilage-like

environment.

Meng

et

al.

found

that

a

tricalcium

phosphate-collagen-hyaluronan scaffold could induce chondrogenic differentiation of human MSCs without any exogenous growth factors.25 Comparatively, we observed that the aBMSC-dECM scaffold also independently induced chondrogenic differentiation. However, the mechanism by which the aBMSC-dECM scaffold induces chondrogenesis remains unclear. One potential option relates to the observation is that cell proliferation is tightly linked to the chondrogenic


Page 18 of 34

18 differentiation of MSCs.26 The aBMSC-dECM scaffold was obtained from autologous BMSCs as well as their ECM components, providing a biocompatible environment for cell adhesion and proliferation. Accordingly, the aBMSC-dECM scaffold might provide an environment conducive to chondrogenic differentiation. A second possibility is that the MSC-derived ECM might protect stem cells from oxidative stress, thereby enhancing proliferation and inhibiting apoptosis.12 Additionally, ECM materials are composed of a complex mixture of molecules that are beneficial to chondrogenic differentiation.27,

28

We also found that several component growth

factors in the aBMSC-dECM scaffold, including bFGF and TGF-β1, might efficiently regulate the chondrogenesis of stem cells (unpublished data). Third, local high cell density might be another factor that induces chondrogenic differentiation.29 It has been reported that direct cell-cell interaction is important for stem cell differentiation, and that low cell density culture exhibits less significant differentiation than high cell density culture.30 Therefore, the high cell density used in our study might also contribute to chondrogenic differentiation.25 Finally, several factors of the scaffold itself, such as pore size, total porosity, pore shape, pore interconnectivity, and limited contraction during culture, are known to influence chondrogenic differentiation. For example, Im et al. reported that chondrogenic differentiation was enhanced in scaffolds with a pore size of 400 µm,31 which is similar to the pore size of the aBMSC-dECM scaffolds used in this study.12 In addition, a more thorough consideration of scaffold size and shape should be made with respect to our results of chondrogenesis ability because of differential scaffold contraction.32 The constructs of


Page 19 of 34

19 the E group maintained their size and shape during in vivo implantation, whereas the volume of the constructs significantly decreased in the C group, suggesting that the aBMSC-dECM scaffolds might be relatively more supportive of chondrogenesis. Another major limitation of cartilage tissue engineering with BMSCs is the lack of stability of the chondrogenic phenotypes. Xue et al. stated that the chondrogenic phenotypes in PGA/PLA scaffolds are lost rapidly when cell differentiation is not completed. The loss of chondrogenic phenotypes appears to accompany increased matrix calcification,33 and PLGA scaffolds seeded with MSCs show matrix calcification after in vivo implantation.34 In our study, when the constructs were cultured with TGF-β3, we found that the E+ group could better resist hypertrophic change, as evidenced by positive staining for sulfate proteoglycan and type II collagen. Compared with the E+ group, the C+ group showed higher expression of COL10A1 during the in vitro culture. However, hypertrophic changes were noted in the outer region of the E group at 3 weeks after in vivo implantation. This might be because the subcutaneous environment is favorable for the invasion of blood vessels that could support hypertrophic change of the construct.35 We also observed that the wall of the aBMSC-dECM scaffold cultured in vivo had completely degraded by 2 weeks. The degradation is too rapid to provide biological function for BMSCs, which is helpless for stabilizing the chondrogenic phenotype. Nonetheless, we also found that the chondrogenic phenotype was maintained at a higher level and in a larger area in the in vivo constructs using the aBMSC-dECM scaffold as compared with the phenotype obtained using other scaffolds such as the PGA/PLA,33 PGA,21 and PLGA scaffolds.34


Page 20 of 34

20 There are some limitations in our study. First, hypertrophic change of the in vivo construct is followed by vessel invasion.36 As our in vivo analysis was based primarily on the expression of chondrogenic and hypertrophic markers, the expression of angiogenic markers during the in vivo implantation should be evaluated as well. Second, time for in vivo implantation was limited to 3 weeks. Although our previous studies showed that the construct in the experiment group could show significant differences compared with that of the control group at 3 weeks after in vivo implantation, limited culture time might lead to some bias in the results.12 Finally, cartilage hypertrophy and calcium deposition were found in the nude mouse model, which differ substantially from the condition of patients with joint disorders. Additional studies will be required to examine and assess the clinical utility of the scaffolds we developed in this work in experimental cartilage defect models and subsequently in patient care.

Conclusions In summary, this study demonstrated that aBMSC-dECM scaffolds can induce chondrogenesis through promotion of the chondrogenic differentiation of BMSCs, and that they could be successful candidate scaffolds for cartilage tissue engineering.

Acknowledgements We thank Professor Hong-Guang Xie, General Clinical Research Center, Nanjing Medical University Nanjing First Hospital, for his critical comments on our


Page 21 of 34

21 manuscript. This research was supported by the National Nature Sciences Foundation of China (81171745).

Disclosure Statement No competing ﬁnancial interests exist.

References 1. Dhollander AA, De Neve F, Almqvist KF, Verdonk R, Lambrecht S, Elewaut D, et al. Autologous matrix-induced chondrogenesis combined with platelet-rich plasma gel: technical description and a five pilot patients report. Knee Surg Sports Traumatol Arthrosc 19, 536, 2011. 2. Hoemann CD, Sun J, McKee MD, Chevrier A, Rossomacha E, Rivard GE, et al. Chitosan-glycerol phosphate/blood implants elicit hyaline cartilage repair integrated with porous subchondral bone in microdrilled rabbit defects. Osteoarthritis Cartilage 15, 78, 2007. 3. Patrascu JM, Freymann U, Kaps C, Poenaru DV. Repair of a post-traumatic cartilage defect with a cell-free polymer-based cartilage implant: a follow-up at two years by MRI and histological review. J Bone Joint Surg Br 92, 1160, 2010. 4. Benthien JP, Behrens P. Autologous matrix-induced chondrogenesis (AMIC). A one-step procedure for retropatellar articular resurfacing. Acta Orthop Belg 76, 260, 2010. 5. Benthien JP, Behrens P. The treatment of chondral and osteochondral defects of


Page 22 of 34

22 the knee with autologous matrix-induced chondrogenesis (AMIC): method description and recent developments. Knee Surg Sports Traumatol Arthrosc 19, 1316, 2011. 6. Schussler O, Shen M, Shen L, Carpentier SM, Kaveri S, Carpentier A. Effect of human immunoglobulins on the immunogenicity of porcine bioprostheses. Ann Thorac Surg 71, S396, 2001. 7. Kim HL, Do JY, Cho HJ, Jeon YC, Park SJ, Ma HI, et al. Dura mater graft-associated Creutzfeldt-Jakob disease: the first case in Korea. J Korean Med Sci 26, 1515, 2011. 8. Lu H, Hoshiba T, Kawazoe N, Chen G. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials 32, 2489, 2011. 9. Zeitouni S, Krause U, Clough BH, Halderman H, Falster A, Blalock DT, et al. Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration. Sci Transl Med 4, 132, 2012. 10. Cai R, Nakamoto T, Kawazoe N, Chen G. Influence of stepwise chondrogenesis-mimicking 3D extracellular matrix on chondrogenic differentiation of mesenchymal stem cells. Biomaterials 52, 199, 2015. 11. Tang C, Xu Y, Jin C, Min BH, Li Z, Pei X, et al. Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering. Artif Organs 37, E179, 2013. 12. Tang C, Jin C, Du X, Yan C, Min BH, Xu Y, et al. An autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold applied with bone marrow stimulation for cartilage repair. Tissue Eng Part A 20, 2455, 2014.


Page 23 of 34

23 13. Pei M, Zhang Y, Li J, Chen D. Antioxidation of decellularized stem cell matrix promotes human synovium-derived stem cell-based chondrogenesis. Stem Cells Dev 22, 889, 2013. 14. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143, 1999. 15. Jin CZ, Cho JH, Choi BH, Wang LM, Kim MS, Park SR, et al. The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect. Tissue Eng Part A 17, 3057, 2011. 16. Jin CZ, Park SR, Choi BH, Park K, Min BH. In vivo cartilage tissue engineering using a cell-derived extracellular matrix scaffold. Artif Organs 31, 183, 2007. 17. Jin CZ, Choi BH, Park SR, Min BH. Cartilage engineering using cell-derived extracellular matrix scaffold in vitro. J Biomed Mater Res A 92, 1567, 2010. 18. Ren Y, Liu B, Feng Y, Shu L, Cao X, Karaplis A, et al. Endogenous PTH deficiency impairs fracture healing and impedes the fracture-healing efficacy of exogenous PTH(1-34). PLoS One 6, e23060, 2011. 19. Meretoja VV, Dahlin RL, Kasper FK, Mikos AG. Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells. Biomaterials 33, 6362, 2012. 20. Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH, Nöth U. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther 9, 213, 2007.


Page 24 of 34

24 21. Choi KH, Choi BH, Park SR, Kim BJ, Min BH. The chondrogenic differentiation of mesenchymal stem cells on an extracellular matrix scaffold derived from porcine chondrocytes. Biomaterials 31, 5355, 2010. 22. Park H, Temenoff JS, Tabata Y, Caplan AI, Mikos AG. Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials 28, 3217, 2007. 23. Bratt-Leal AM, Carpenedo RL, Ungrin MD, Zandstra PW, McDevitt TC. Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation. Biomaterials 32, 48, 2011. 24. Pagnotto MR, Wang Z, Karpie JC, Ferretti M, Xiao X, Chu CR. Adeno-associated viral gene transfer of transforming growth factor-beta1 to human mesenchymal stem cells improves cartilage repair. Gene Ther 14, 804, 2007. 25. Meng F, He A, Zhang Z, Zhang Z, Lin Z, Yang Z, et al. Chondrogenic differentiation of ATDC5 and hMSCs could be induced by a novel scaffold-tricalcium phosphate-collagen-hyaluronan without any exogenous growth factors in vitro. J Biomed Mater Res A 102, 2725, 2014. 26. Dexheimer V, Frank S, Richter W. Proliferation as a requirement for in vitro chondrogenesis of human mesenchymal stem cells. Stem Cells Dev 21, 2160, 2012. 27. McDevitt CA, Wildey GM, Cutrone RM. Transforming growth factor-beta1 in a sterilized tissue derived from the pig small intestine submucosa. J Biomed Mater Res A 67, 637, 2003. 28. Hodde JP, Badylak SF, Brightman AO, Voytik-Harbin SL. Glycosaminoglycan


Page 25 of 34

25 content of small intestinal submucosa: a bioscaffold for tissue replacement. Tissue Eng 2, 209, 1996. 29. Zhang L, Yuan T, Guo L, Zhang X. An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells. J Biomed Mater Res A 100, 2717, 2012. 30. Tang J, Peng R, Ding J. The regulation of stem cell differentiation by cell-cell contact on micropatterned material surfaces. Biomaterials 31, 2470, 2010. 31. Im GI, Ko JY, Lee JH. Chondrogenesis of adipose stem cells in a porous polymer scaffold: influence of the pore size. Cell Transplant 21, 2397, 2012. 32. Zhang L, Yuan T, Guo L, Zhang X. An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells. J Biomed Mater Res A 100, 2717, 2012. 33. Xue JX, Gong YY, Zhou GD, Liu W, Cao Y, Zhang WJ. Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells induced by acellular cartilage sheets. Biomaterials 33, 5832, 2012. 34. Mehlhorn AT, Zwingmann J, Finkenzeller G, Niemeyer P, Dauner M, Stark B, et al. Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A 15, 1159, 2009. 35. Steck E, Fischer J, Lorenz H, Gotterbarm T, Jung M, Richter W. Mesenchymal stem cell differentiation in an experimental cartilage defect: restriction of hypertrophy to bone-close neocartilage. Stem Cells Dev 18, 969, 2009. 36. Noël D, Gazit D, Bouquet C, Apparailly F, Bony C, Plence P, et al. Short-term


Page 26 of 34

26

BMP-2 expression is sufficient for in vivo osteochondral differentiation of

mesenchymal stem cells. Stem Cells 22, 74, 2004.

Figure legends

Tissue Engineering Part A Chondrogenic differentiation could be induced by autologous bone marrow mesenchymal stem cell–derived extracellular matrix scaffolds without exogenous growth factor (doi: 10.1089/ten.TEA.2014.0491) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 27 of 34

27

Fig. 1 Gross observation and volume measurement of the in vitro constructs after 3,

10 and 21 days. (A) Gross observation of the constructs cultured with (C+ and E+

group) or without (C- and E- group) TGF-β 3. (B) Volume measurement of the

constructs in each group. Results were presented as means ± SD.


Page 28 of 34

28

Fig. 2 Accumulation of sulfate proteoglycan of the in vitro constructs after 3, 10 and

21 days. (A) The constructs in each group were stained with safranin O. Sulfate

proteoglycan positive areas during in vitro culture (B) and after 21 days (C) were

measured by computer-assisted image analysis. Results were presented as means ±

SD. ＊P < 0.05. A: ×10 and 400.


29

Fig. 3 Accumulation of type Ⅱ collagen of the in vitro construct after 3, 10 and 21

days. (A) The constructs in each group were analyzed by immunohistochemical

staining for type II collagen. Type Ⅱ collagen positive areas during in vitro culture

(B) and after 21 days (C) were measured by computer-assisted image analysis.

Results were presented as means ± SD.

＊

P < 0.05. A: ×10 and 400.


Page 30 of 34

30

Fig. 4 Gene express of the in vitro constructs after 3, 10 and 21 days. The expression

value of cartilage matrix-related genes, including COL2A1 (A), ACAN (B), SOX9 (C),

COL1A2 (D) and COL10A1 (E), were calculated relative to GAPDH expression.

Results were presented as means ± SD.


31

Fig. 5 Total DNA content (A and a), GAG content (B and b) and GAG/DNA ratio (C

and c) of the in vitro constructs after 3, 10 and 21 days. Results were presented as

means ± SD. ＊P < 0.05.


Page 32 of 34

32

Fig. 6 Gross observation and volume measurement of the in vivo constructs. (A)

Gross observation of the constructs in the C and E groups. (B) Volume measurement

of the constructs. Results were presented as means ± SD. ＊

P < 0.05, ＊＊＊

P < 0.001.


33

Fig. 7 Expression of chondrogenic markers of the in vivo constructs. (A) The

constructs in each group were analyzed by safranin

Results were presented as means ± SD. ＊P < 0.05. A: ×400. O staining and

immunohistochemical staining for type II collagen. Sulfate proteoglycan (B) and type

Ⅱ collagen (C) positive area were measured by computer-assisted image analysis.


Page 34 of 34

34

Fig. 8 Expression of hypertrophic markers of the in vivo constructs. (A) The

constructs in each group were analyzed by Micro-CT imaging and Von Kossa staining.

Calcified matrix volume (B) and calcified mineral deposits area (C) were analyzed

quantitatively. Results were presented as means ± SD.

＊

P < 0.05. A: ×10 and 400.

Preparation and characterization of bone marrow mesenchymal stem cell-derived extracellular matrix scaffolds.

Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering.

Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro.

Influence of stepwise chondrogenesis-mimicking 3D extracellular matrix on chondrogenic differentiation of mesenchymal stem cells.

Icariin promotes directed chondrogenic differentiation of bone marrow mesenchymal stem cells but not hypertrophy in vitro.

Long noncoding RNAs expression signatures in chondrogenic differentiation of human bone marrow mesenchymal stem cells.

MicroRNA-410 promotes chondrogenic differentiation of human bone marrow mesenchymal stem cells through down-regulating Wnt3a.

Response of endothelial cells to decellularized extracellular matrix deposited by bone marrow mesenchymal stem cells.

Angiogenesis induced by autologous whole bone marrow stem cells transplantation.

cartilage extracellular matrix scaffolds with sequential delivery of TGF-β3 for chondrogenic differentiation of adipose-derived stem cells.

Nerve growth factor from Chinese cobra venom stimulates chondrogenic differentiation of mesenchymal stem cells.

Determination of the chondrogenic differentiation processes in human bone marrow-derived mesenchymal stem cells genetically modified to overexpress transforming growth factor-β via recombinant adeno-associated viral vectors.

Laminin matrix promotes hepatogenic terminal differentiation of human bone marrow mesenchymal stem cells.

Autologous bone-marrow mesenchymal cell induced chondrogenesis (MCIC).

PLLA scaffolds.

Cell Fate and Differentiation of Bone Marrow Mesenchymal Stem Cells.

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells.

Characteristics and response of mouse bone marrow derived novel low adherent mesenchymal stem cells acquired by quantification of extracellular matrix.

Dynamic nanomechanics of individual bone marrow stromal cells and cell-matrix composites during chondrogenic differentiation.

Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow-derived mesenchymal stem cells.

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium.

Neural differentiation of mouse bone marrow-derived mesenchymal stem cells treated with sex steroid hormones and basic fibroblast growth factor.

Rapid growth and osteogenic differentiation of mesenchymal stem cells isolated from human bone marrow.

Cell type-specific extracellular matrix guided the differentiation of human mesenchymal stem cells in 3D polymeric scaffolds.