Hydrodynamic loading in concomitance with exogenous cytokine stimulation modulates differentiation of bovine mesenchymal stem cells towards osteochondral lineages
© Goldman and Barabino. 2016
Received: 13 May 2015
Accepted: 18 January 2016
Published: 1 February 2016
Mesenchymal stem cells (MSCs) are viewed as a having significant potential for tissue engineering and regenerative medicine therapies. Clinical implementation of MSCs, however, demands that their preparation be stable and reproducible. Given that environmental and bioprocessing parameters such as substrate stiffness, seeding densities, culture medium composition, and mechanical loading can result in undirected differentiation of the MSC population, the objective of this study was to systematically investigate how hydrodynamic loading influences the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) towards the osteochondral lineages both in the presence and absence of exogenous, inductive factors.
Expanded bovine MSCs were suspended in 2.5 % agarose, cast in a custom mold, and placed into either static or one of two dynamic culture environments consisting of “high” and “low” magnitude shear conditions. Constructs were supplemented with varying concentrations (0, 1, 10, 100 ng/mL) of either TGF-β3 or BMP-2 throughout cultivation with tissue samples being collected following each week of culture.
In the absence of exogenous supplementation, hydrodynamic loading had little effect on cell phenotype at either magnitude of stimulation. When cultures were supplemented with BMP-2 and TGF-β3, MSCs gene expression progressed towards the osteogenic and chondrogenic pathways, respectively. This progression was enhanced by the presence of hydrodynamic loading, particularly under high shear conditions, but may point the chondrogenic cultures down a hypertrophic path toward osteogenesis reminiscent of endochondral ossification if TGF-β3 supplementation is insufficient.
Moving forward, these results suggest bioprocessing conditions which minimize exposure of chondrogenic cultures to fluid shear stress to avoid undesirable differentiation of the MSC population.
KeywordsMesenchymal stem cells Fluid shear stress Chondrogenesis Osteogenesis Bioprocessing Biomanufacturing
Due to their limited supply and decreased proliferative capacity, the sole use of autologous chondrocytes and osteoblasts for regenerative medicines is likely unsustainable . Subsequently, mesenchymal stem cells (MSCs) have emerged as a clinically relevant cell source for regenerative medicine, due to their ease of procurement, multipotentiality, high proliferation rate, and ability to be expanded in vitro while maintaining a stable phenotype [2–4]. Directed differentiation of MSCs along various mesenchymal pathways can be achieved by manipulation of the cell culture environment including supplementation of culture medium with soluble morphogens [5–9], modulation of culture substrate stiffness , and external forces [11, 12]. Of particular interest are environmental approaches which might increase differentiation efficiency while reducing upstream bioprocessing costs for the purpose of large scale commercial operations.
A predominant challenge of the scaling operations required to process large numbers of cells and/or critically sized tissue constructs is the control of nutrient and waste transport from the cells/tissues during culture. To overcome these issues, a number of bioreactor concepts have been developed to provide the flow of culture media through , across , and around the constructs [15, 16]. As a result of the medium exchange, the constructs are concurrently nourished and exposed to hydrodynamic loading. Shear stress is known to cause varied effects on cell populations, including transmembrane ion leakage, as well as physiological and metabolic changes . The presence of fluid shear stress, therefore, is an important environmental factor which may play an important role in the stability or instability of the MSC phenotype in culture. Furthermore, if the magnitude and spatiotemporal presentation of hydrodynamic loading can be controlled, it may represent a novel approach to modulating the efficiency of directed MSC differentiation. The primary objective of this study, therefore, was to determine the effect of uniform shear stress magnitude and duration on MSC gene expression through a panel of key differentiation markers along the osteochondral differentiation pathway. These genes were selected for their importance in orthopedic tissue engineering applications and potential to provide a window into the chondrogenic and osteogenic differentiation processes.
Given the well-documented sensitivity of mature chondrocytes and osteoblasts to the TGF-β superfamily , a secondary objective of this study was to examine the response of MSCs to varying magnitudes of superficial hydrodynamic shear stress in cultures supplemented with varying concentrations of TGF-β3 and BMP-2. Drawing on evidence that both bone and cartilage are mechanosensitive [18, 19] and mechanical stimuli are anabolic [20, 21], we hypothesized that hydrodynamic loading would increase the efficiency of MSC differentiation down the desired pathways as revealed through systematic changes in phenotypic markers. The scope of the study was limited to a range of hydrodynamic conditions within the reported interstitial flow regime of bone  and cartilage  with a view to determining an optimum for lineage specific differentiation. Additionally, the cytokine concentrations were varied by one order of magnitude in either direction from the most ubiquitous supplementation protocols found in the literature concurrently, to determine how hydrodynamic culture might minimize their necessity.
Unless specified otherwise, supplies and reagents were purchased from VWR International (West Chester, PA), Sigma (St. Louis, MO) or Invitrogen (Carlsbad, CA). Antibodies were from AbD Serotec (Raleigh, NC) or Abcam (Cambridge, MA).
To elucidate the role of hydrodynamic loading in MSC differentiation towards the osteochondral lineages, we selected three magnitudes of fluid shear stress (0, 1, 10 dyn/cm2) to be applied in the presence of four levels of exogenous stimulation (0, 1, 10, 100 ng/mL) for two different cytokines (BMP-2, TGF-β3) resulting in 12 experimental groups which received some level of both hydrodynamic and exogenous stimuli, three groups which received only TGF-β3 stimulation of varying degrees, three groups which received only BMP-2 stimulation of varying degrees, and three unsupplemented groups which received only hydrodynamic stimulation of varying degrees (Additional file 1: Table S1). Samples from each experimental group were collected on a weekly basis for 2 weeks. Additional samples for each group were generated at the start of tissue culture (Week 0), but never subjected to any of the stated experimental conditions in order to generate a baseline for downstream analysis.
Endogenous controls were evaluated for each experimental group to ensure that their expression levels were not significantly altered across time or culture conditions.
For histological analysis, constructs were fixed in 10 % buffered formalin, embedded in paraffin and sectioned into 8 μm thick sections for the midsubstance of the construct. Sectioned samples were stained with Toluidine blue and Alizarin Red per established protocols. For immunofluorescence, sections were incubated with a citrate buffer heated to 99 °C for 30 min to retrieve antigens, and allowed to cool to room temperature. The samples were then incubated in blocking buffer for 30 min and primary rabbit anti-bovine antibodies (1:100, Abcam, Cambridge, MA) for Collagen types I, II, and X at 4 °C overnight. Sections were then washed three times in PBS and with DyLight®594 goat anti-rabbit secondary antibodies (1:200, Abcam, Cambridge, MA) for one hour at room temperature. Finally, samples were washed and mounted with Vectashield with DAPI and visualized on a Nikon Ti Eclipse inverted fluorescence microscope (Nikon Instruments, Inc., Melville, NY), with representative images captured using a CoolSNAP HQ2 CCD camera (Photometrics, Tucson, AZ).
Independent experiments produced construct samples for RT-qPCR and immunohistochemistry (N = 3 per group). Gene expression is presented as the mean fold change ± SEM with statistically significant differences defined as p <0.05 using two-way ANOVA with Bonferroni post-hoc tests for multiple comparisons.
Stability of gene expression in unsupplemented cultures
Effect of hydrodynamic loading on unsupplemented cultures
Effect of cytokine supplementation on differentiation markers
While hydrodynamic loading in isolation of exogenous supplementation is not sufficiently potent to control differentiation in a selective manner, the results of our initial studies in unsupplemented, serum-free cultures suggested that hydrodynamic loading may be useful as when presented in concert with morphogens with a known inductive capacity. To investigate this possibility, we analyzed the expression profiles of statically cultured constructs which received either TGF-β3 or BMP-2 supplementation at a concentration of 1, 10 ng/mL, or 100 ng/mL for the purpose of inducing a chondrogenic or osteogenic phenotype, respectively. The resident MSCs tend towards expression of chondrogenic markers as function of time in culture and concentration of exogenous cytokine supplementation for both BMP-2 and TGF-β3 supplementation.
Effect of hydrodynamic loading on cytokine supplemented cultures
Both BMP-2 and TGF-β3 produced strong differentiation of the MSC population utilizing the static culture platform, and provided a baseline for normalization of the hydrodynamically loaded cultures to control for the independent effect of the cytokines so that we might investigate whether stimulation via hydrodynamic loading can induce a synergistic effect on the gene expression profile of the differentiating cell population.
Regarding the chondrogenic markers, SOX9 was upregulated in high magnitude loading culture relative to the static control for all BMP-2 supplementation groups, but interestingly this effect was only considered significant at the 1-week time point. COL2A1 expression was observed to increase in high magnitude hydrodynamic cultures as well, as evidenced by significant increases relative to static controls at the 2-week time point for cultures receiving 1 ng/mL of BMP-2 and at both time points for culture receiving at least 10 ng/mL of BMP-2. This effect also appears to be sensitive the magnitude of hydrodynamic loading as significant differences were observed between the static cultures and low magnitude cultures as well as between the low and high magnitude cultures. Differences in AGGRECAN expression were only considered significant under high shear and high supplementation. It is also worth noting that expression of hypertrophic marker COLXA1 was significantly increased in the high magnitude loading group after 2 weeks of culture for all BMP-2 supplementation protocols relative to the static control for the low concentrations (1 ng/mL) and to both low magnitude and static cultures at elevated concentrations of BMP-2 (10 and 100 ng/mL).
These results are not very surprising in light of the results from unsupplemented, hydrodynamically loaded construct group as two osteoinductive agents, hydrodynamic loading and BMP-2 supplementation, are at work simultaneously in these protocols. While the slight chondrogenic character of these cultures is not desirable, it is worth noting that modulation of chondrogenic markers (SOX9, COL2Α1) at high shear was of less than an order of magnitude and the order of the baseline control expression of these genes was considerably lower than their chondrogenic counterparts. COLXΑ1 expression increased by an order of magnitude over culture period and supplementation matched static controls for both low and high shear conditions at two weeks when cultures were supplemented with 100 ng/mL of BMP-2. While the inductive impact of hydrodynamic loading is not as great in magnitude as that of BMP-2 supplementation at high levels (one order of magnitude change vs three orders of magnitude), it none the less is an important modulator of osteogenic induction as no significant difference was observed between static cultures supplemented at 100 ng/mL and cultures supplemented at 10 ng/mL that were also subjected to high magnitude hydrodynamic loading in terms of total gene expression relative to the initial MSC population.
Interestingly, changes in expression of the osteogenic gene panel were not considered significant for any of the hydrodynamic regimes studied. Expression of COLXΑ1, however, was upregulated relative to concentration matched static controls when conditions were such that high shear magnitudes (10 dyn/cm2) were paired with low (1 ng/mL) concentrations of TGF-β3 for a period of at least 2 weeks. No significant changes in COLXA1 were observed with moderate or high TGF-β3 supplementation. These observations hint at two potentially useful characteristics of this approach. First, chondrogenic differentiation is clearly positively influenced by the presence of hydrodynamic loading when presented in concert with at least 10 ng/mL of TGF-β3, and that TGF-β3 signaling appears to have an inhibitory effect the on the osteoinductive role of high magnitude hydrodynamic loading observed with the other supplementation protocols studied herein.
Histology & immunofluorescence
As the predominate source of cells for tissue engineered constructs has shifted from terminally differentiated primary cells towards progenitor cells of various differentiation potentials, the ability to spatiotemporally exert epigenetic control over the differentiation of stem cells within a tissue engineered construct has become desirable as a means to better reproduce native tissue complexity and reduce cultivation costs associated with traditional differentiation protocols. Subsequently, we decided to focus herein on the impact of the hydrodynamic environment on MSC differentiation due to its essential role in nutrient exchange during in vitro cultivation. The purpose of this study was to investigate how modulation of hydrodynamic loading affects the stability of the MSC phenotype during serum-free tissue culture and how this approach might enhance differentiation efficiency in the presence of morphogens known to be either osteoinductive or chondrogenic in nature. Our primary findings (Additional file 1: Table S3) were that hydrodynamic loading, in the absence of exogenous supplementation, promotes expression of hypertrophic and osteogenic genes. Although not investigated in this study, we suspect this observation is due to integrin signaling activated by deformation of the MSC pericellular matrix in response to the hydrodynamic loads as indicated by other studies in the literature [30, 31]. The influence of hydrodynamic loading on osteogenic markers was sustained for low levels of exogenous supplementation irrespective of the cytokine provided. Lineage specific upregulation towards chondrogenic and osteogenic phenotypes was observed under high magnitude shear conditions when hydrodynamic loading was presented in concert with high levels of TGF-β3 and BMP-2 supplementation respectively. Generally, the observed impact of hydrodynamic loading on the desired phenotypes was greater in longer term cultures, and in cultures receiving higher concentrations of exogenous cytokines.
These findings are in agreement with prior mechanobiological studies in other osteochondral lineage cell sources. In studies based on osteoblastic cell lines, multiple studies have shown that hydrodynamic loading is osteopromotive [32–36], and often results in increases in type I collagen production and matrix mineralization. Hydrodynamic studies on MSCs from various donor species have also been previously shown to be osteoinductive [37–39]. Additionally, multiple studies on primary chondrocytes have shown that in addition to increases in type II collagen [40, 41]. Exposure to high shear environments can result in development of a fibrous layer rich in non-hyaline type I collagen at shear exposed surfaces , particularly when cultured with serum supplemented medium  versus serum free preparations. If we compare the extent of the impact of hydrodynamic loading on unsupplemented cultures in our study to that of other environmental induction schemes for MSCs, we find that the effect on gene expression is on the same order of magnitude as manipulations of scaffolding stiffness for osteoinduction  and both hydrostatic pressure  and dynamic unconfined compressive loading [46, 47] for chondrogenic induction. Unlike these prior studies, however, we found the impact of exogenous supplementation on gene expression to be considerably greater than the environmental stimulus applied. Our results converge again, however, when the mechanical stimuli were presented concurrently with TGF-β3 supplementation [45, 46]. As in our study utilizing hydrodynamic loading, dynamic compression and intermittent hydrostatic loading both resulted in additional increases in chondrogenic gene expression when presented in cultures supplemented with at least 10 ng/mL. The order of magnitude of the change, however, is considerably greater in our hydrodynamic study (>100 fold change) than either of the prior studies utilizing compressive (<10 fold change) and hydrostatic (<10 fold change) loading.
Conversely, other studies have shown compressive loading to have a negative impact on glycosaminoglycan accumulation within the construct at the protein level . It is unclear, however, if this effect is due to decreased synthesis or loss of glycosaminoglycans to the culture medium. Interestingly, the same study , also showed that dynamic compressive loading resulted in increases in COLXA1 in the absence of TGF-β3 supplementation, a result that mirrors our findings of both a hypertrophic influence of unsupplemented hydrodynamic loading and of the chondroprotective character of TGF-β3 supplementation. Our findings, herein, seem to indicate a comparable role of hydrodynamic loading to that of other environmental factors, particularly dynamic compression. Considering finite element analyses have shown interstitial fluid flow to be an effect of dynamic loading in biphasic materials such as those referenced herein, it is not surprising that these two loading conditions produce similar responses in MSC based tissue constructs.
Our finding that high magnitude hydrodynamic loading promotes osteogenic gene expression in unsupplemented cultures is instructive and suggests that MSCs cultures intended for chondral therapies not be subjected to high shear hydrodynamic loading conditions during processing and cultivation. While the modified parallel plate bioreactor system utilized better utilized for experimental investigation than a scalable manufacturing process, the bioprocessing principles derived from its use could easily be incorporated into a more scalable suspension bioreactor system based on three dimensional microcarriers. It is our recommendation that the nutrient utilization of chondrogenic cultures in such a system be carefully considered such that fluid loading not be applied in excess of magnitudes needed to meet the convective transport demands of the tissue in order to avoid potential induction of a hypertrophic phenotype. Conversely, our findings also suggest hydrodynamic loading of osteogenic cultures can potentially be a means of either reducing culture dependence on exogenous cytokines or promoting increased matrix deposition provided the magnitude of loading is increased such that impacts cell viability in a negative manner .
The findings of this study bring forth a number of important considerations regarding hydrodynamic culture of MSC based constructs for tissue engineering applications. As evidenced by results from all cytokine supplementation groups, including serum-free expansion medium culture, it is clear that MSCs are tuned to their local mechanical loading environment, and that prolonged exposure to high magnitude fluid shear stresses induces a hypertrophic phenotype amongst the resident MSCs ultimately resulting in expression of osteogenic markers. For the purpose of chondrogenic cultures, therefore, our results suggest minimizing the fluid shear stress imposed on the developing construct without reducing the transport of nutrients to all regions of the tissue construct. Furthermore, this phenomenon presents an interesting paradigm for the production of osteochondral tissue constructs through differential loading of the construct, both chemically and hydrodynamically, by varying the microenvironment appropriately in spatially separated regions of the tissue construct. While the overall goal of the current study of a single medium source with differential loading to induce phenotypic changes in the MSC population was not achieved, there is evidence that loading will play a significant role in bioprocessing protocols of osteochondral constructs moving forward as technologies such as microfluidic hydrogels [50–53] provide the means to differentially apply chemical and environmental cues within an integrated construct of a single cell type to spatially engineer osteochondral tissues for intra-articular injury repair and preclinical models for pharmacological studies against osteoarthritis.
No acknowledgements to report.
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