Decellularized Articular Cartilage Microparticles for Expansion of Mesenchymal Stem Cells and Zonal Regeneration of Articular Cartilage

Introduction The objective was to create multilayer cellular constructs using fetal or adult, decellularized articular cartilage in particulate form as microcarriers for expansion and fusion of mesenchymal stem cells (MSCs) to regenerate the stratified structure of articular cartilage. Methods Porous microparticles (CMPs) generated from decellularized fetal or adult bovine articular cartilage were used as microcarriers for expansion of human MSCs. The CMP expanded MSCs (CMP-MSCs) were used to generate injectable hydrogels or preformed multilayer constructs for articular cartilage regeneration. In the injectable approach, CMP-MSCs were suspended in alginate gel, crosslinked with calcium chloride, and incubated in chondrogenic medium to generate an injectable regenerative construct. In the preformed approach, fetal or adult CMP-MSCs were suspended in a culture medium, allowed to settle sequentially by the force of gravity, and fused by incubation in chondrogenic medium to generate multilayer cell sheets. The constructs were characterized with respect to compressive modulus, cellularity, and expression of chondrogenic markers. Results Human MSCs expanded on fetal or adult CMPs in basal medium maintained the expression of mesenchymal markers. The injectable CMP-MSCs hydrogels had significantly higher expression of chondrogenic markers and compressive modulus after four weeks incubation in chondrogenic medium compared to MSCs directly encapsulated in alginate gel; preformed CMP-MSCs cell sheets had significantly higher compressive modulus and expression of chondrogenic markers compared to MSCs in the pellet culture. Conclusion The preformed cell sheet approach is potentially useful for creating multilayer constructs by sequential gravitational settling of CMP-MSCs to mimic the stratified structure of articular cartilage. Insight, Innovation, Integration This work described a novel approach to recreate the zonal structure of articular cartilage. Human MSCs were expanded on porous microcarrier beads generated from decellularized fetal or adult bovine articular cartilage. The cell-seeded microbeads were fused by gravitational settling to form mono- or bi-layer cell sheets. The cell sheets were cultured in chondrogenic medium to regenerate the articular cartilage tissue. The in vitro regenerated tissue had higher compressive modulus and expression of chondrogenic markers compared to the MSC pellet culture.


Introduction
Cartilage degeneration is prevalent in older adults with 65% of those over 60 years old experiencing joint pain and long-term disability [1][2][3]. Due to its complex physical and biochemical properties, there has been limited clinical success in treatment of articular cartilage injuries [4]. Autologous Chondrocyte Implantation (ACI) is currently used in the clinic to treat cartilage injuries. Although ACI shows better long-term outcomes than microfracture technique or other traditional methods [5,6], its success is limited by poor cell retention, non-homogenous cell distribution, dedifferentiation of implanted cells, periosteal hypertrophy, and donor site morbidity [7,8]. Thus, alternative methods have been explored for the repair of cartilage defects.
A promising alternative to chondrocyte harvesting is the use of adult human mesenchymal stem cell, hereafter referred to as MSCs, derived from the bone marrow or synovium [9,10].
MSCs delivered in a supportive matrix have been shown to promote the expression of chondrogenic markers and produce a cartilage-like matrix in vivo. However, the approach of MSC encapsulation in a uniform matrix, without gradients, often leads to fibrocartilage formation and tissue degeneration [11,12]. The formation of inferior fibrocartilage tissue is rooted in the inability of cellular constructs to recapitulate the stratified structure of articular cartilage [13]. There is clearly a need for novel engineering approaches to recreate the zonal structure of full-thickness articular cartilage defects without fibrocartilage tissue formation.
The stratified structure of articular cartilage is composed of the superficial, middle, deep and calcified zones with each zone having a defined protein expression, cellularity, extracellular matrix (ECM) composition and structure for lubrication, compressive strength, and load transfer to the subchondral bone [14]. It is well established that fetal articular cartilage, with a stratified structure from week 12 of gestation, has a higher regenerative capacity compared to the adult, mainly due to difference in cellularity and ECM composition [15]. Engineering approaches that mimic cellularity, ECM composition and structure of fetal articular cartilage could enhance regeneration of full-thickness articular cartilage defects.
We previously demonstrated for the first time that high cellularity, low matrix stiffness and combination of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7) led to chondrogenic differentiation of MSCs to the superficial zone phenotype of articular cartilage; medium cellularity and stiffness, and combination of TGF-β1 and IGF-1 led to the middle zone phenotype; and low cellularity, high matrix stiffness and combination of TGF-β1 and hydroxyapatite (HA) led to the calcified zone phenotype [16,17]. Further, we recently demonstrated that MSCs encapsulated in digested, decellularized articular cartilage could sequentially be differentiated to the superficial zone phenotype, followed by the middle and calcified zones by sequential supplementation of the chondrogenic medium with BMP-7, insulin growth factor-1 (IGF-1) and Indian hedgehog (IHH) [18]. In addition, we showed that digested and decellularized fetal articular cartilage induced differentiation of MSCs to the superficial zone phenotype of chondrocytes whereas the adult cartilage induced differentiation to the calcified phenotype.
Biodegradable microcarriers, due to their large surface area, have been used as 3D matrices for expansion of stem cells [19,20]. Commonly used microcarriers, aside from triggering undesirable phenotypic changes, require additional processing steps to separate the expanded cells from the carrier for clinical applications. We hypothesized that MSCs attached to developmentally inspired, articular cartilage microparticles (CMPs) can mimic the process of fetal development when combined with zone-specific growth factors to regenerate the stratified structure of articular cartilage [21]. Multilayer constructs with gradients in cell density, matrix composition, and morphogens can be assembled with from MSCs attached to CMPs, hereafter referred to as CMP-MSCs, for articular cartilage regeneration.
The following approach, illustrated schematically in Figure 1, was used to test the hypothesis. Fetal or adult bovine articular cartilage was decellularized, milled in liquid nitrogen, and the freeze-dried fragments were grinded and sorted to generate fetal (fCMPs) or adult (aCMPs) CMP fractions of different average sizes. Next, the fetal or adult MSCs were used as microcarriers for expansion of MSCs to generate fetal or adult CMP-MSCs, respectively. Then, an injectable and a prefabricated approach were used to generate articular cartilage tissues. In the injectable approach, fetal or adult CMP-MSCs were encapsulated in alginate hydrogel, hereafter referred to as CMP-MSCs/alg, and cultured in chondrogenic medium to form cartilage tissues. In the prefabricated approach, fetal or adult CMP-MSCs were suspended in chondrogenic medium,

Production of decellularized bovine cartilage microparticles
Full thickness articular cartilage samples, harvested from fetal or adult bovine femoral condyles, were decellularized as we described previously. Briefly, the articular cartilage samples were dissected with a scalpel into 5x5x2 mm pieces, the dissected pieces were frozen in liquid nitrogen and milled. The milled fragments were decellularized by immersion in 10 Mm Tris/1% triton solution for 24 h followed by sonication for 2 h at 55 kHz. Next, the sonicated fragments were immersed in nuclease solution consisting of 1 U/mL deoxyribonuclease and 1 U/mL ribonuclease in PBS for 72 h at 37°C to degrade DNA and RNA [22]. The decellularized fragments were washed 3X in PBS, centrifuged, the supernatant was discarded, and the solid was freeze-dried.
The freeze-dried fragments were further grinded (Hamilton Beach, Southern Pines, NC) and sorted for size by progressively passing through sieves ranging in size from 80 to 300 µm. First, the soft fragments were passed through 80 and 300 µm sieves to eliminate the <80 µm and >300 µm fragments. Next, the soft, decellularized cartilage microparticles (CMPs) were passed through a 90 µm sieve to collect a fraction with 40-110 µm size range, which is referred to as the 90 µm CMPs. Next, the >90 µm CMPs were passed through a 190 µm sieve to collect a fraction with 60-220 µm size range, which is referred to as the 190 µm CMPs. Then, the >190 µm CMPs were passed through a 250 µm sieve to collect a fraction with 60-300 µm size range, which is referred to as the 250 µm CMPs. The above process was repeated for the >250 µm fraction to sort the particles into the three 90, 190 and 250 µm fractions. Fetal and adult CMPs are hereafter referred to as fCMPs and aCMPs, respectively.

2.3.Characterization of the decellularized articular cartilage microparticles
Microparticle size distribution: The sieved CMPs were imaged with a light microscope to determine their size distribution. The captured 2D images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD) as we described previously [23]. After size analysis, the fetal and adult CMP fractions were immersed in liquid nitrogen and cut with a surgical blade to expose a freshly cut surface for morphological analysis. Next, the CMPs were coated with gold using a Denton Desk II sputter coater (Moorestown, NJ) at 20 mA for 75 s. The CMPs were imaged with a TESCAN VEGA3 SBU variable-pressure scanning electron microscope (SEM; Kohoutovice, Czech Republic) at an accelerating voltage of 8 keV.

Measurements of water content and mass loss:
The equilibrium fractional water content of the dried, decellularized CMPs was measured by incubation in phosphate buffer saline (PBS) at 37°C as we described previously [24]. Briefly, after swelling in PBS, the CMPs were filtered, unbound water was removed with a filter paper, and weight of the samples was measured. After weighing, the samples were returned to fresh PBS solutions and incubated until the next time point. This process was repeated until equilibrium swelling was achieved. Equilibrium water content of the CMPs was calculated as the difference between the initial and swollen weights divided by the total weight of CMPs. Mass loss of the CMPs samples was measured at one time point after 8 weeks of incubation in PBS. After 8 weeks, the CMPs were filtered, freeze-dried, and weight of the samples was measured. Mass loss was defined as the difference between the initial and final weights of the dry CMPs divided by the initial dry weight.

Culture of MSCs on articular cartilage microparticles
MSCs (passage 3-5) were expanded in a high glucose DMEM medium supplemented with 10% FBS, 100 units/mL penicillin G, and 100 µg/mL streptomycin (basal medium, BM) as we previously described [18]. CMPs were sterilized by immersion in ethanol for 2 h followed by exposure to ultraviolet (UV) radiation for 30 min [25]. After washing, the sterilized adult or fetal CMPs were incubated in basal culture medium overnight to swell prior to cell seeding. The initial seeding density was calculated based on the average size of MSCs (18 µm average diameter [26]) and size and surface area of the CMPs. The initial cell seeding density was based on 1x10 6 MSCs occupying 20% of the surface area of CMPs (20% initial confluency). For seeding 3x10 6

Characterization of MSCs cultured on articular cartilage microparticles
Cell viability and growth: At each time point, 2 mL of 0.05% trypsin/ 0.53 mM EDTA was added to 1 mL of the cell culture suspension and incubated for 15 min under shaking to detach MSCs from the CMPs. Next, the suspension was transferred to a 40 µm nylon cell strainer fixed on a 50 mL Falcon tube and washed with DMEM using an insulin syringe. The filtrate was centrifuged at 400×g for 5 min as we described previously [28], and the separated cells were counted with a hemocytometer [28]. For cell viability, the CMPs were stained with 1 µg/mL live/dead cAM/EthD and imaged with an inverted fluorescent microscope (Nikon Eclipse Ti-e, Nikon, Melville, NY), as we described previously [24].
Analysis of mRNA expression: The MSCs cultured on CMPs were characterized phenotypically by mRNA upregulation of CD105, CD166 and CD44 markers, and downregulation of CD45 and CD34 markers [29]. Further, the MSCs cultured on CMPs were tested for differentiation to the chondrogenic lineage by measuring mRNA expression of chondrogenic markers SOX-9, Collagen I (Col I), Collagen II (Col II), and aggrecan (AGC) [17,18]. At each time point, MSCs were separated from the CMPs as described above and the total RNA of the homogenized cell suspension was isolated using TRIzol as we described previously [23]. The genomic DNA was removed using deoxyribonuclease I (Invitrogen) as previously described [16]. 250 ng of the extracted RNA was converted to cDNA using Promega reverse transcription system (Madison, WI). The cDNA was amplified with Eppendorf SYBR green RealMasterMix (Hamburg, Germany) using a Bio-Rad CXF96 real-time quantitative polymerase chain reaction system (rt-qPCR; Hercules, CA) and the appropriate gene-specific primers as descried [17]. The primer sequences, listed in Table 1, were designed and selected using Primer3 web-based software as described [16]. The expressions were normalized against GAPDH reference gene and fold changes were compared based on ∆∆ct method to those in the same group at day zero as previously described [30]. Table 1 2

.6.Encapsulation of CMP-MSCs in alginate as an injectable hydrogel
The following approach was used to generate CMP-MSC encapsulated alginate gels as an injectable cell carrier for articular cartilage regeneration [31]. The alginate (3 g sodium alginate to 100 mL PBS) and CaCl2 (1 g CaCl2 to 100 mL PBS) solutions [25] were sterilized by filtration. A suspension of adult or fetal CMP-MSCs in culture medium, prepared as described in section 2.4, was transferred to a sterile 15 mL Falcon tube, centrifuged, medium was removed, and the alginate solution was added to the CMP-MSCs followed by mixing with a pre-sterilized glass rod. Next, the surface of a pre-sterilized, disk-shape, Teflon mold with effective diameter of 2 cm and height of 1.5 mm was sprayed with the 1% CaCl2 solution using an insulin syringe.
Then, the suspension of CMP-MSCs in sodium alginate was transferred to the mold and the

Formation of CMP-MSC monolayer and bilayer cell sheets
The following approach was used to generate CMP-MSC cell sheets as a preformed cell carrier for articular cartilage regeneration. A suspension of adult or fetal CMP-MSCs (approximately 50% cell confluency) in culture medium, prepared as described in section 2.4, was transferred to a sterile Teflon mold (1.55 mm depth and 2 cm in diameter), the mold was placed in a sterile petri dish, and the assembly was incubated in a humidified 5% CO2 incubator at 37°C for 48 h. Gravitational settling of the CMP-MSCs on the bottom surface of the mold followed by ECM secretion led to the formation of adult or fetal CMP-MSCs monolayer cell sheets, hereafter referred to as aCMP-MSCs/ml or fCMP-MSCs/ml cell sheet, respectively. The CMP-MSC loading in the culture medium was varied to form cell sheets with 0.75 mm thickness. The following approach was used to produce bilayer cell sheets. After formation of aCMP-MSC/ml, a suspension of fCMP-MSCs was transferred to the mold with aCMP-MSC/ml cell sheet, and the assembly was incubated for 48 h to gravitational settle fCMP-MSCs on top of aCMP-MSCs/ml cell sheet followed by ECM secretion to form a bilayer cell sheet, hereafter referred to as faCMP-MSCs/bl cell sheet. The CMP-MSC loading in the culture medium was adjusted to form faCMP-MSCs/bl cell sheets with 1.5 mm thickness. After cell sheet formation, the medium was replaced with chondrogenic medium and the cell sheets were cultured for up to 8 weeks. The chondrogenic medium consisted of DMEM (4.5 g/mL glucose, 50 μg/ mL Lproline, 50 μg/mL ascorbic acid, 0.1 mM sodium pyruvate, 1% v/v insulin-transferrin-selenium premix) supplemented with 10 ng/mL TGF-β1 [16]. MSC pellets formed directly by centrifugation were used as the control group [32].

Analysis of monolayer, bilayer, or alginate encapsulated CMP-MSCs
At each time point, adult or fetal CMP-MSCs/alg, CMP-MSCs/ml or CMP-MSCs/bl were assessed with respect to compressive modulus, cellularity, and the expression of chondrogenic markers Sox-9, Collagen II (Col II) and aggrecan (AGC), the superficial zone marker SZP, and calcified zone markers collagen X (Col X) and alkaline phosphatase (ALP). Cell viability was assessed by imaging with live/dead cell assay. The mono/bi-layer sheets were incubated with cAM/EthD live/dead stains, as we described previously [16], and the stained samples were imaged using an Eclipse Ti-E inverted fluorescent microscope. For cell imaging, CMP-MSCs/alg gels were fixed with 4% paraformaldehyde for 3 h, permeabilized using PBS containing 0.1% Triton X-100 for 5 min, and incubated with Alexa 488 phalloidin (1:200 dilution) and DAPI (1:5000 dilution) to stain actin filaments of the cell cytoskeleton and cell nuclei, respectively, as previously described [33]. The stained gels were imaged with the Eclipse inverted fluorescent microscope. The mRNA expression of chondrogenic markers of MSCs in the samples were measured as described in section 2.5.

Histological Analysis
After 21 days of culture, the expression of GAG and mineralized deposits in aCMP-MSCs/ml, fCMP-MSCs/ml, or faCMP-MSCs/bl was measured histologically as we previously described [16]. Briefly, the samples were fixed in formalin, embedded in paraffin, and cryosectioned to a thickness of 10 µm. The sections were divided into three groups with the first group stained with H&E to ascertain morphology of the encapsulated cells, the second group stained with Alcian blue to image GAG accumulation, and the third group stained with Alizarin red to image mineral deposit. The stained sections were imaged with a Nikon Optiphot Epifluorescent microscope. and subjected to a uniaxial compressive strain as we previously described [34]. A strain sweep was performed from 0.01% to 500% strain at 10 Hz to determine the yield strain. Similarly, a frequency sweep was performed from 0.01 to 100 Hz at 0.2% strain to determine the crossover frequency. A sinusoidal shear strain with a frequency above the crossover frequency and a strain amplitude below the yield strain was exerted on the sample and the storage (G') and loss moduli (G") were recorded with time. The slope of the linear fit to the stress-strain curve for strains of <10% was taken as the compressive modulus of the samples.

Statistical Analysis
All experiments were done in triplicate and quantitative data was expressed as means + standard deviation. Significant differences between groups were evaluated using a two-way ANOVA with replication test and two-tailed Student's t-test. A value of p > 0.05 was considered statistically significant. of particles with size greater than the average was higher for fCMPs compared to aCMPs, which was attributed to the softer texture of fCMPs and their passage through the sieve with a slight pressure. The harder texture of aCMPs resulted in a smaller particle size distribution compared to the fetal. The CMPs had irregular, non-spherical shapes and there was no difference in the shape of fetal and adult CMPs.
The mass loss of fetal and adult CMPs increased with increasing particle size. For a given particle size, the mass loss of aCMPs was slightly lower than fCMPs. The mass loss data indicated that the fetal and adult CMPs are stable for up to 8 weeks in the absence of enzymes with negligible hydrolytic degradation.  CMPs was attributed to higher inter-particle cell transfer with each successive CMP addition to the culture medium. Inter-particle transfer requires cell detachment from one particle, migration through the medium, and re-attachment to another particle with lower cell content, which increased the lag time between cell divisions. Figure 3c compares the cell content of fetal and adult CMPs as a function of average particle size with incubation time. For a given time and particle size, the cell content of fCMPs was slightly higher than aCMPs but the difference was not statistically significant.

Figure 3
The live (green) and dead (red) fluorescent images of a randomly selected particle in fetal or adult CMP samples are shown in Figure 4 as a function of incubation time for different average sizes. Based on fluorescent images, the MSCs penetrated the pore structure of CMP particles.
The intensity of green fluorescence from the CMPs increased with incubation time for fetal as well as adult CMPs and for all particle sizes. After 21 days of incubation, the fluorescent intensity of fCMPs was slightly higher than aCMPs for all particle sizes but the difference was not statistically significant. The fluorescent images of CMP-MSCs showed >95% cell viability.

Figure 4
According to previous reports, the expression of CD105 and CD44 in human MSCs harvested from the bone marrow is upregulated whereas the expression of CD45 and CD34 is down regulated [35,36]. Further, the as-received MSCs had high expression of CD105, CD166,  CaCl2 concentration. For a given CaCl2 concentration, gelation time increased with increasing CMP particle size. Figure 6b shows the effect of CMP loading on gelation time of the alginate solution for different concentrations of CaCl2. For a given CaCl2 concentration, the gelation time decreased with increasing CMP loading from 30% to 70% by volume. In general, the gelation times were in the range of 1-4 min, which were within the clinically acceptable range for injectable cell-encapsulated hydrogels [38].

Figure 6
The graphs in exhibit A of Figure 7 compare mRNA expression of chondrogenic markers for adult or fetal CMP-MSCs/alg hydrogels as a function of incubation time in chondrogenic medium. The control group in exhibit A was MSCs encapsulated directly, without CMPs, in alginate. Chondrogenic markers included Sox-9 [37], SZP as the superficial zone marker [39], Col II and AGC as the middle zone markers [16], and Col X and ALP as calcified zone markers [40]. For all markers and incubation times, adult or fetal CMP-MSCs showed higher expressions compared to directly encapsulated MSCs in alginate. There was no difference between the expressions of Sox-9, AGC, Col X and ALP of aCMP-MSCs/alg and fCMP-MSCs/alg whereas SZP was higher in aCMP-MSCs/alg and Col II was lower. As the culture medium was not supplemented with zone-specific growth factors, we did not expect a significant difference in the expression of zone-specific markers between aCMP-MSCs and fCMP-MSCs, consistent with our previous results that matrix composition and zone-specific growth factors work synergistically to enhance MSC differentiation to zone-specific chondrogenic phenotypes [16]. According to the data in exhibit A of Figure 7   The graphs in exhibit B of Figure 7 compare mRNA expression of chondrogenic markers for aCMP-MSCs/ml, fCMP-MSCs/ml, and faCMP-MSCs/bl as a function of incubation time in chondrogenic medium. The control group in exhibit B is the commonly used 3D pellet culture [32]. For Sox-9, SZP and AGC markers, the expressions for CMP-MSCs monolayers and bilayers were relatively close to the pellet culture whereas for Col II, Col X and ALP makers, the expressions for CMP-MSCs were higher than the pellet culture. There was not a difference in marker expressions between the adult and fetal CMP-MSCs cell sheets, except for Col II of fCMP-MSCs/ml which was slightly higher than faCMP-MSCs/bl. The data in Figures 5-7 show that the MSCs expanded CMPs could potentially be injected or implanted in an articular cartilage defect without the need to detach and separate MSCs from CMPs. Further, the experimental results indicate that the adult or fetal CMPs, as a biomimetic microcarrier, enhance chondrogenic differentiation and maturation of MSCs.