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Responses of MSCs to 3D Scaffold Matrix Mechanical Properties under Oscillatory Perfusion Culture Guobao Chen, Rui Xu, Chang Zhang, and Yonggang Lv ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10745 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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ACS Applied Materials & Interfaces

Responses of MSCs to 3D Scaffold Matrix Mechanical Properties under Oscillatory Perfusion Culture Guobao Chen, †,‡,§,# Rui Xu, §,ơơ,# Chang Zhang, ơơ Yonggang Lv *,†,‡,§ †

Postdoctoral Research Station of Biology, Chongqing University, Chongqing 400044, P. R. China ‡

Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044, P. R. China §

Mechanobiology and Regenerative Medicine Laboratory, Bioengineering College, Chongqing University, Chongqing 400044, P. R. China

ơơSchool

of Environmental Engineering, Wuhan Textile University, Wuhan 430073, P. R.

China KEYWORDS: matrix stiffness, 3D scaffold, mesenchymal stem cells, perfusion culture, bioreactor, bone tissue engineering

ABSTRACT: Both fluid shear stress and matrix stiffness are implicated in bone metabolism and functional adaptation, but the synergistic action of these mechanical cues on the biological behaviors of mesenchymal stem cells (MSCs) is still not well known. In the present work, a home-made oscillatory flow device was applied to investigate the effects of matrix stiffness on MSCs survival, distribution and osteogenic differentiation in

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three-dimensional (3D) conditions. Furthermore, the flow field and cell growth in this bioreactor were theoretically simulated. The results demonstrated that oscillatory shear stress significantly increased the viability and distribution uniformity of MSCs throughout the scaffold after culture for 3 weeks. Compared to static culture, oscillatory shear stress could promote the collagen secretion, mineral deposits, and osteogenic differentiation of MSCs. The findings obtained from this work indicate that the oscillatory perfusion not only provides a higher survival rate and a more uniform distribution of cells, but also facilitates osteogenic differentiation of MSCs. Oscillating perfusion bioreactor culture of MSCs in 3D scaffold with optimal matrix stiffness could offer an easy-to-use but efficient bioreactor for bone tissue engineering.

1. INTRODUCTION It is well-accepted that mechanical stimulation can enhance bone tissue regeneration in vitro, with lots of researches proving that osteogenic differentiation of osteoprogenitor cells can be enhanced by optimal mechanical force and physiological stimulation.1 In vivo, bone-tissue cells can sensitively feel and respond to alterations of mechanical cues in their mechanical microenvironment. The mechanical microenvironment of bone cells is a dynamic environment of biomechanical stimuli applied on bone cells, mainly including strain, matrix mechanics, fluid shear stress, pressure, and so on.2 Among the variety of biophysical stimuli, many in vitro studies have demonstrated that matrix stiffness and


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fluid shear stress play a critical role in directing the activity of osteoprogenitor cells. On the one hand, the extracellular matrix (ECM) also appears to have a vital role in maintaining and remodeling bone tissue in vivo.3 For instance, the stiffness of bone increases with increasing mineralization and organization of the matrix, maturation of bone crystals, and replacement of woven bone by lamellar bone.4 Furthermore, matrix mechanical cues can regulate the osteogenic differentiation of osteoprogenitor cells5,6 and the bone remodeling process.7,8 For example, the stiffness of the substrate defined the lineage commitment of the human mesenchymal stem cells (MSCs): the soft brain-like substrates (0.1-1 kPa) were found to induce the neurogenic differentiation, intermediate stiffness muscle-like substrates (8-17 kPa) were found to induce myogenic differentiation, and relatively stiff substrates (25-40 kPa) could induce the osteogenic differentiation.5 Recently, the effects of matrix stiffness of three-dimensional (3D) scaffold on the behavior of stem cells have gotten researchers’ much attention due to the remarkable difference between two-dimensional (2D) and 3D microenvironments.9,10 Major obstacles in studying the impact of matrix mechanics in 3D are short of usable engineering scaffolds in which the stiffness can be adjusted without relying on the matrix density.11 To overcome this obstacle,

our group has successfully fabricated novel 3D scaffolds with

same microstructure but different matrix stiffness, in which collagen (Col)/hydroxyapatite (HA) mixture in different collagen ratio was used to coat decellularized bone scaffold to obtain 6.74±1.16 kPa, 8.82±2.12 kPa, and 23.61±8.06 kPa matrix stiffness.12 Our in vitro and in vivo experimental studies12,13 have found these scaffolds can sustain the MSCs



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adhesion, growth, and osteogenic differentiation and enhance the new bone formation. On the other hand, a heterogeneous cell distribution and cell death occurrence in the center are still major obstacles for culturing the 3D tissue-engineered scaffold for long time in static condition in vitro due to the mass transport limitation.14 The limitations of nutrient delivery, oxygen supply and exchange, and metabolic-waste discharge eventually decrease the survival rate of cells inside the scaffold, form non-uniform cellular distribution within scaffold, and limit the usable size of 3D scaffold. To overcome these drawbacks associated with static culturing system, the most common approach is to use a bioreactor system to generate a dynamic environment for cell-loaded scaffold culture.15 The bone tissue bioreactor systems not only provide a more uniform nutrient supply in the 3D construct, but also apply different shear stress stimulation to cells.16 Bone is predominately subjected to oscillating fluid flow and unidirectional flow in vivo, which represent two major patterns of flow in human daily activities.17 Different posture changes may cause different flow patters. For instance, human body change from sitting to standing forms unidirectional flow, while walking and running (typical cyclic activities of skeletal system) always generates oscillating fluid flow.18 Hence, oscillatory shear stress is much closer to the physiological pattern than unidirectional shear stress.19 Previous studies implied that oscillatory fluid flow can promote the proliferation of osteoblast-like cells,20 stimulate the Ca2+ release and osteopontin (OPN) gene expression of MC3T3-E1 osteoblasts,21 and regulate the intracellular communication among osteocytic MLO-Y4 cells.22 These studies suggested that oscillatory fluid flow can


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positively impact the cell-cell interactions and enhance osteogenic differentiation of osteoprogenitor cells. Although both fluid shear stress and matrix stiffness are implicated in bone metabolism and functional adaptation, it is not yet known whether these mechanical cues can affect the lineage fate determination of MSCs synergistically. Here, we investigated the cooperative effects of 3D matrix stiffness and oscillatory shear stress on the MSCs survival, distribution, collagen secretion, and osteogenic differentiation. The fluid shear stress distribution and cell growth in this bioreactor were theoretically calculated by numerical simulation method.

2. MATERIALS AND METHODS 2.1. Scaffold Fabrication. Scaffolds were treated as previously described12 and some improvements were made. To ensure their uniformity, the porcine cancellous bone scaffold was obtained from the same position of femoral head in pig. Col/HA mixture with different collage ratios (Col 0.35/HA 22, Col 0.5/HA 22, and Col 0.7/HA 22) was coated on decellularized bone scaffolds to form different matrix stiffness (6.74±1.16 kPa, 8.82±2.12 kPa, and 23.61±8.06 kPa).12 More specifically, collagen was dissolved in 0.01 M HCl solution at different concentrations (0.35, 0.5, and 0.7 wt %). And then HA power was added into the collagen solutions to form 22 wt % mixtures of HA.12 All scaffolds were prepared as cylindrical shape (diameter: 10 mm; height: 5 mm) to fit for the chambers of perfusion system.

60

Co γ irradiation (25 k Gay) (Allanace, The Third



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Military Medical University, Chongqing, China) was used to sterilize the scaffolds before use.

2.2 Oscillatory Perfusion Bioreactor System. Rat MSCs were exposed to oscillatory shear stress using a 3D oscillatory perfusion bioreactor system.23 The 3D oscillatory perfusion bioreactor system was fabricated in our laboratory as shown in Figure S1. In brief, the perfusion bioreactor system consists 4 independent experimental chambers and each chamber (diameter: 10 mm; height: 8 mm) is able to accommodate one scaffold. The top hole in the chamber was sealed tightly with a screw top. Culture medium perfused the 3D porous scaffold through the screw top and entered the media reservoir connected with the bottom of the chamber. The media was supplied by a syringe pump (PHD2200, Harvard Apparatus, USA) at 1 mL/min. Based on the previous studies,24,25 the oscillatory frequency was set as 1/60 Hz in the present study. To avoid the occurrence of gas bubble in the bioreactor during perfusion, the culture medium should be regulated to ensure no gas bubbles. The system was assembled in a cell culture hood to maintain sterility.

2.3 MSCs Seeding and Culture. Primary rat bone marrow-derived MSCs were isolated from four-week-old Sprague-Dawley (SD) rats weighing 130 ± 10 g by flushing their femurs and tibias and further cultured and identified following the method outline in our previous publication.26 All animal work were approved by Chongqing University Animal Care and Use Committee. Cells were expanded in at 37 ºC with 5% CO2 in low


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glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (16000-044, Gibco, USA) to the third passage, and used for the following experiments. 1.6×106 MSCs in 40 µL of DMEM were pipetted into the top of the sterilized scaffolds. In order to obtain a relatively uniform distribution of seeded cells, the cell-loaded scaffold was flipped 4 times in 1 h and then cultured in an incubator (3308, Thermo Scientific, USA). After culture for 2 days, the scaffolds (4 per group) were transferred into the chambers of oscillatory perfusion bioreactor system as the perfusion culture groups for 30 min per day.27,28 In order to avoid the pollution in long-term cultivation process, after oscillatory perfusion for 30 min per day, the treated scaffolds were transferred into the 6-well plate and further cultured. The oscillatory perfusion bioreactor system was cleaned and sterilized for the next time using. The static culture group was cultured in 6-well plate for the same time. Culture medium in each group was changed every other two days.

2.4 Live/dead Assay. All scaffolds were taken out from the chambers and cut longitudinally along its midline. One half was used for live/dead staining following the instructions of the manufacturer (LIVE/DEAD Viability/Cytotoxicity Kit, Molecular Probes, USA). The living and dead cells were labeled with calcein AM (green) and ethidium homodimer-1 (red), respectively. All images were taken from the middle of the scaffolds and five fields from each sample were obtained under the microscope (Olympus



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IX71, Japan). No less than three samples for each group were evaluated by live/dead assay.

2.5 Micro-computed Tomography (µ-CT). µ-CT was performed by using the method from Grayson et al.29 The scaffold was stabilized with sponge in a 50 mL centrifuge tube and placed into a specimen holder using µ-CT (Scanco medical AG vivaCT 40, Switzerland). All scaffolds were scanned longitudinally at 19.5 µm isotropic resolution, before and after perfusion culture and static culture for 1 week. 2D and 3D images were reconstructed by the software to investigate the porosity, wall thickness and so on.30

2.6 Hematoxylin-eosin (H-E) and Masson's Trichrome Staining. After culture for 1, 2, and 3 weeks, all cell-scaffold constructs were fixed using 4% paraformaldehyde for 3 days and then decalcified by 12% EDTA-2Na (A500838, Sangon Biotech, China) for about 1 month. The decalcified bone scaffold was dehydrated in ethanol and embedded by paraffin to make 7-µm longitudinal sections using a microtome (RM 2235, Leica, Germay). The paraffin sections were further performed with H-E staining (C0105, Beyotime, China) and Masson’s trichrome staining (D026, Nanjing Jiancheng Biotechnology Institute, China). The samples were analyzed under a light microscope (Olympus IX71, Japan).

2.7 Immunohistochemistry. Endogenous peroxidase activity of paraffin-embedded


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section was blocked by 0.3% hydrogen peroxide for 20 min (SP9001, IHC staining module, Beijing Zhongshan Biotechnology, China). After blocking of nonspecific protein binding sites with bovine serum albumin (BSA), the anti-rat osteocalcin (OC) antibody (bs0470R, Bioss, China; diluted 1:50) and anti-rat OPN antibody (ab8448, Abcam, USA; diluted 1:300) were added overnight at 4 ºC. Then, the application of secondary antibody was accomplished according to the manufacturer’s instructions (SP9001, IHC staining module, Beijing Zhongshan Biotechnology, China). After diaminobenzidine (DAB) coloration, section was washed and coverslipped. Sample analyses were carried out three separate times under using a light microscopy (Olympus IX71, Japan). For semiquantitative analysis of OPN- and OC- positive expression, the average IOD per area (IOD/area) was estimated for each section by using the Image-Pro Plus software 6.0 (Media Cybernetics, USA).

2.8. Mathematical Model. The theoretical model was assumed as a sandwich-like model, in which a scaffold was fixed between and two fluid layers at the top and bottom. Fluid was pumped into the scaffold through the entrance region of the inlet fluid layer. The symmetry plane of the perfusion bioreactor was sketched out in Figure 1, in which x is the radial direction and y denotes the axial direction of the bioreactor. Following assumptions were made to simplify the solution of theoretical equation: (1) Our previous study indicated that the scaffold has good interconnectivity with typical porous structure.12 The scaffold layer is treated as a two-phase medium, in which α phase



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denotes the culture medium while β phase comprises the deposited ECM, scaffold and adherent cells on the scaffold. (2) The culture medium is Newtonian and incompressible. Flow inlet

L 1

H

Fluid layer

3

2

4

H

Scaffold layer

6

5

7

H

Fluid layer

9

8 α phase

y

β phase

10 0

x

Flow outlet Figure 1. Schematic representation of the perfusion system. A sandwich-like model was consisted of a scaffold and two fluid layers. The enlarged are denotes the elementary volume which comprises α phase (culture medium) and β phase (scaffold, cell adhering to the scaffold, and deposited cell secretion). x denotes the distance from the boundary of the cylinder in radial direction and y are along the centerline in axial direction.


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(3) The culture temperature was maintained at 37 oC with negligible effects on the properties of culture medium. (4) The degradation of the scaffold is very small during the perfusion culture and neglected in the simulation. All the pores in the scaffold are assumed to be interconnected. (5) There is no floating cell in the culture medium. The density change of the culture medium due to nutrient consumption and metabolic waste accumulation is very small. The culture medium density was assumed as a constant. (6) As an important nutrition, glucose was used as the representative nutrient in our simulation.31 Nutrient transports in the α phase through diffusion and convection. Transports in the β phase by diffusion. (7) Ignoring the MSCs differentiation, the volume of solid phase (i.e. β phase) increases and the porosity of the scaffold decreases during perfusion culture because of cell proliferation and secretion. Briefly, the effect of fluid shear stress on the MSCs growth was simulated by the modified Contois cell-growth kinetics.32 A typical convection-diffusion model was used to describe the nutrient distribution in the two fluid layers. In the scaffold layer, the nutrient consumption was governed by Michaelis-Menton equation.31 The Navier-Stokes equations and Brinkman’s equation were used to model the fluid flow in the two fluid layers and scaffold layer, respectively. In the fluid layer, the fluid velocity u can be calculated by



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∇⋅u = 0

(1)

T ∂  u +( u ⋅ ∇)u  = −∇P + µ∇ ⋅ ∇u + ( u )     ∂t 

ρ ⋅

(2)

where, ρ is the density of culture medium, t the culture time, µ the dynamic viscosity of the culture medium, and P is the pressure. In the scaffold layer, the fluid velocity can be calculated by

∂ε + ∇ ⋅ ub = 0 ∂t

ρ

(3)

∂ ( ub ⋅ ε ) T µ = −∇P − ( ub ⋅ ε ) + µ∇ ⋅ ∇ ( ub ⋅ ε ) + ( ∇ ( ub ⋅ ε ) ) ∂t κ

(

)

(4)

where, ε is the porosity of the scaffold field, ub the local fluid velocity vector in the scaffold layer, κ the permeability of the construct, which is solved by Carman-Kozeny equation33

κ = K p ε 3 (1 − ε )

2

(5)

where, Kp is a reference value. For nutrient transfer equations, the nutrient concentration in the fluid layer can be obtained by ∂cα = ∇ ⋅ ( Dα ∇cα ) − ∇ ⋅ ( cα u ) ∂t

(6)

where, cα is the nutrient concentration in the culture medium, Dα is the diffusion coefficient of glucose in α phase and assumed constant value during the culture time. The nutrient concentration in the scaffold layer can be obtained by

∂ ( ε + σ β ⋅ K eq ) cα  ∂t

= ∇ ⋅ ( Deff ∇cα ) − ∇ ⋅ ( cα ub ) −

Rm cα ⋅ (σ β − σ s ) K m + cα

(7)

The last term on the right side of Equation (7) used the Michaelis-Menten kinetics to


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calculate the nutrient consumption by cells. σβ=1-ε is the volume fraction of β phase in the fluid layer, σs is the volume fraction of scaffold. Then the volume fraction of cells in the fluid layer is represented by (σβ-σs). Rm is the maximum metabolic rate of glucose consumption for seeded cells, Km is the saturation coefficient. Keq is the equilibrium coefficient. Deff is the effective diffusivity for glucose in the scaffold layer, which can be solved by Maxwell’s formula34

Deff = Dα

3ω − 2ε (ω − 1) 3 + ε (ω − 1)

(8)

where, ω = K eq Dβ Dα , Dβ is the diffusion coefficient of glucose in cell. Considering the nutrient equilibrium on the interface of α and β phases, the nutrient concentration in cells cβ can be obtained by35

cβ = K eq cα

(9)

The cellular growth was derived from the modified Contois kinetics35 ∂ (σ β − σ s ) ∂t

 Rg cα = Dcell ∇ 2 (σ β − σ s ) +  −1 − Rd + Qτ  K eq K c ρcell (σ β − σ s ) + cα 

  (σ β − σ s ) (10)  

where, Dcell is the diffusion coefficient of cell random motion, Rg is cell maximum specific growth rate, Kc is the modified Contois saturation constant, ρcell is the mass density of single cell, Rd is the death rate of cells. Qτ represents the effect of local shear stress on cellular growth32 Qτ = aτ 0.3 + bτ

(11)

Based on the results from Li et al.,36 the values of a and b are 1×10-6 and -1.5×10-6, respectively. Shear stress τ is calculated as



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 ∂ub ∂vb  +   ∂y ∂x 

τ = µ

(12)

where, ub and vb are the flow rate along x and y directions in the scaffold layer, respectively. The average cell volume fraction in a porous scaffold field was defined as

σβ −σs = ∫

L

x=0

∫ (σ 2H

y=H

β

− σ s ) dydx

L

2H

x =0

y=H

∫ ∫

dydx

(13)

The boundary and initial conditions for the above governing equations in the Supporting Information. The values of all parameters involved in the governing equations were cited from the references and provided in Table S1. The mathematical model was solved by COMSOL Multiphysics. Mesh quality were tested to keep the calculation convergence discrepancy smaller than 10-3. In order to validate the computational program used in this study, the program was used to calculate the equations proposed in previous study.31 The comparison results (Figure S2) could prove that the computational program was reliable.

2.9 Statistical Analysis. All experiments were performed at least three times. Data were expressed as the mean ± standard deviation (SD). Results were compared respectively through the one-way analysis of variance (ANOVA). p-value < 0.05 was considered as statistically significant; * represents p < 0.05.

3. RESULTS


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3.1 Prediction of the Distribution of Velocity, Glucose Concentration and Cell Volume Fraction in the Scaffold by Theoretical Model. Theoretical model developed in this study was used to calculate the distribution of velocity, glucose concentration and cell volume fraction in the scaffold made in our previous study.12 Figure 2 showed the transient average cell volume fraction under different flow patterns. As time goes on, both unidirectional flow and oscillatory flow dramatically increase the average cell volume fraction in the scaffold. With the same total volume flux, the unidirectional flow had the best efficiency for cell growth. Comparing to the static culture condition, unidirectional flow and oscillatory flow increased the average cell volume fraction from 4.57% to 32.18% and 29.99% after culture for about 20 days, respectively. With the same total volume flux, unidirectional flow may supply more nutrition and faster waste remove in the scaffold. Figure 3 further showed the distribution of velocity, glucose concentration, and cell volume fraction in the scaffolds at 7 days, 14 days, and 21 days. The distribution of cell volume fraction under static culture condition was different from those under unidirectional flow and oscillatory flow. The cell volume fraction close to the center (y=0.75 cm) was lower than those near the fluid layers (y=0.5 cm and y=1.0 cm). On the other hand, under unidirectional flow and oscillatory flow, the cell volume fraction close to the boundary (x=0) was higher than that near the center (x=0.5 cm). At the beginning of culture, the glucose concentration under oscillatory flow was lower than that under unidirectional flow. However, there was no difference for glucose concentration among them after culture for 21 days.



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Figure 2. Transient average cell volume fraction under unidirectional flow (u0=600 µm/s), oscillatory flow (u0=30+300πsin(2πt) µm/s) and without flow (u0=0) (ε=85%).


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Figure 3. Distribution of velocity, glucose concentration, and cell volume fraction in the scaffolds at 7 (A), 14 (B), and 21 days (C) (ε=85%, unidirectional flow: u0=600 µm/s, oscillatory flow: u0=30+300πsin(2πt) µm/s, without flow: u0=0).



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3.2 Oscillatory Perfusion Increases the Survival and Distribution Uniformity of Cells throughout the Scaffold. Live/dead assay was performed after culture for 1, 2, and 3 weeks to evaluate the influences of oscillatory perfusion on the cell survival and distribution on different scaffolds (Figure 4). Our previous work12 showed that the most living MSCs mainly adhere to the scaffold’s surface and distributed inhomogeneous under static condition. Compared with the static culture,12 the living cells distributed more uniformly throughout the scaffold under dynamic culture. Lots of living cells with green fluorescence were observed on the pore surfaces of the scaffolds (Figure 4A). Meanwhile, all the scaffolds contained a very small amount of dead cells with red fluorescence. Quantitative data of dead cells per field of the scaffold indicated that the number of dead cells remained at relatively low level during the culture process (Figure 4B). These results demonstrated that most of the cells within the 3D porous scaffolds were viable after culture for 3 weeks under oscillatory flow. Compared with culture for 1 week under oscillatory flow, the cell numbers increased at 2 and 3 weeks. Moreover, areas near the scaffold surfaces had the highest cell density under oscillatory condition.


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Figure 4. Live/dead assay of the midline section of different scaffolds seeded with MSCs for 1, 2, and 3 weeks under oscillatory perfusion. (A) Geen shows the living cells and red indicates the dead cells. The scale bar indicates 50 µm. (B) Quantitative data of dead cells per field of the scaffold at different time points. Control indicates the decellularized bone scaffold.

3.3 Oscillatory Perfusion Increases the Mineral Content of MSCs in Different Scaffolds. The accumulation and maturation of bone-like tissue were confirmed by µ-CT



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imaging before and after culture for 1 week to observe the influences of oscillatory perfusion on the mineral content of cells in different scaffolds (Figure 5). In the Col 0.7/HA 22 group, pore size and wall thickness of the scaffold were 382.8 µm and 108.5 µm before perfusion. After perfusion culture for 1 week, the mean pore diameter and wall thickness of the scaffold were 370.2 µm and 116.4 µm (Table 1). The results showed that oscillatory perfusion increased the mineralized tissue portion and the wall thickness of scaffolds, along with decrease in pore size of scaffolds.

Figure 5. Reconstructed 3D µ-CT images of the scaffolds after culture for 1 week in oscillatory perfusion bioreactor. Control indicates the decellularized bone scaffold. The scale bar indicates 2 mm.

Table 1. Comparison of pore size and wall thickness in different scaffolds before and after oscillatory perfusion culture. Control

Col 0.35/HA 22 Col 0.5/HA 22 Col 0.7/HA 22

Wall thickness (µm) Before perfusion Pore size (µm)

102.9

113.2

103.9

108.5

445.4

359.8

345.7

382.8

Wall thickness (µm) After perfusion Pore size (µm)

130.0

131.1

123.5

116.4

393.8

252.4

310.8

370.2


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3.4 Oscillatory Perfusion Promotes the ECM Secretion and Deposition of MSCs in Different Scaffolds. Figure 6 showed the H-E staining of tissue deposition in the midline section of the scaffolds cultured with MSCs for 1, 2, and 3 weeks under oscillatory perfusion. Compared to the static culture for 1 week, oscillatory fluid shear stress increased the number of cells in the middle of the scaffolds. Furthermore, oscillatory perfusion culture has a more homogenous deposition of ECM compared with static culture, especially at week 3.

Figure 6. H-E staining of tissue deposition in the midline section of the scaffolds cultured with MSCs for 1, 2, and 3 weeks under the oscillatory perfusion (the black arrow indicates typical deposition of ECM). Control indicates the decellularized bone scaffold. The scale bar indicates 100 µm.

3.5 Oscillatory Perfusion Promotes the Collagen Secretion of MSCs in Different Scaffolds. Figure 7 showed the Masson trichrome staining of collagen deposition in the



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midline section of the scaffolds cultured with MSCs for 1, 2, and 3 weeks under oscillatory perfusion. After static culture for 1 week, the top row of Figure 7 showed a gradual increase in collagen staining (blue color) in scaffolds along a continuous stiffness gradient. In contrast to static culture, oscillatory perfusion cultures had deeper staining of collagen (blue color) in the same culture period and same stiffness. Meanwhile, representative histological sections from the scaffolds with different stiffness in the oscillatory perfusion bioreactor showed a positive correlation between collage deposition and matrix stiffness. These results indicated that the oscillatory perfusion and matrix stiffness may synergistically affect the collagen secretion of cells on the scaffolds.

Figure 7. Masson trichrome staining of collagen deposition in the midline section of the scaffolds cultured with MSCs for 1, 2, and 3 weeks under oscillatory perfusion. Control indicates the decellularized bone scaffold. The scale bar indicates 100 µm.


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3.6 Oscillatory Perfusion Culture Enhances the Osteogenic Differentiation of MSCs in Scaffolds with Different Matrix Stiffness. To observe the osteogenic differentiation of MSCs in different groups, The expression levels of OPN and OC were detected by immunohistochemistry on histological sections at 1, 2, and 3 weeks to observe the osteogenic differentiation of MSCs under oscillatory perfusion culture (Figure 8). After culture for 1 week under static culture, the expression levels of OPN and OC in all three coating groups showed a slight increase than those in the control group. The positive staining of OPN and OC is much higher in Col 0.7/HA 22 group than other groups. After oscillatory perfusion bioreactor culture for 2 weeks, the expression of OPN and OC in Col 0.7/HA 22 exhibited a higher level than other groups (Figure 8). After oscillatory perfusion bioreactor culture for 3 weeks, the highest expression levels of OPN and OC were appeared in the scaffolds with optimal matrix stiffness (Col 0.7/HA 22) than others (Figure 8). Compared to static culture, the oscillatory perfusion could promote the osteogenic differentiation of MSCs after culture for the same time. These results indicated that the oscillatory perfusion and matrix stiffness may have a synergistic action on the osteogenic differentiation of MSCs in the scaffolds.



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Figure 8. Immunohistochemistry staining of OPN (A) and OC (B) in the midline section of the scaffolds cultured with MSCs for 1, 2, and 3 weeks under oscillatory perfusion. The scale bar indicates 100 µm. Quantitative data for expression of OPN (C) and OC (D) in the midline section of the scaffolds cultured with MSCs for 1, 2, and 3 weeks under oscillatory perfusion. Control indicates the decellularized bone scaffold. S: static; P: perfusion. * indicates p < 0.05.

4. DISCUSSION It is very important to develop a high performance system to increase the osteogenic ability of the cell-scaffold construct, besides, a workable 3D culture platform for keeping the viability of cell-scaffold construct after prolonged culture in vitro. In addition, the traditional 2D culture platform and the 3D culture system for stem cell differentiation


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may work differently. In the present work, the cooperative action of 3D matrix stiffness on MSCs differentiation by oscillatory perfusion culture was studied by using a home-made 3D oscillatory flow device to apply oscillatory perfusion on the scaffolds. Taken together, our results demonstrated a strong interplay between oscillatory shear stress and matrix mechanics that affects MSCs survival, distribution, mineralization, collagen secretion, and osteogenic differentiation. Cell survival and uniformity of cell distribution in 3D scaffolds are usually the first consideration in vitro or in vivo studies. It is quite important to develop a local microenvironment similar to natural condition. However, the traditional static culture easily caused the death of seeded cells in 3D scaffolds. In order to overcome these limitations, dynamic 3D perfusion bioreactor can be designed to supply the adequate nutrients and oxygen for seeded cells in vitro. Sikavitsas et al.37 found that the spinner flask culture can enhance proliferation of cells at the first and second week compared to static condition. However, the cell proliferation phenomenon did not appear in the third week. In another study, the 3D dynamic spinner flask culture did not have significant survival improvements compared to those in the static culture conditions during week one.38 After 3 weeks, a 20% DNA content was increased by dynamic versus static culture. The differences of cell proliferation curve between the two reports may be related to the various sources of MSCs. In the current study, the computational fluid dynamic modeling confirmed that unidirectional and oscillatory flow dramatically increases the average cell volume fraction in the construct. Unidirectional flow had a slight higher cell growth than



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oscillatory flow. Furthermore, the importance of oscillatory flow in osteogenesis has been confirmed in many axial perfusion bioreactors by in vitro experiments.39 Oscillatory flow culture only need a small amount of culture medium, which can reduce the cost of drugs or proteins in the culture medium.40 Thus, the oscillatory flow was selected in the current study. In the present study, the result of live/dead staining demonstrated that the oscillatory perfusion significantly improved the distribution of living cells through the whole 3D scaffold. The homogeneously distribution of MSCs throughout the 3D scaffold maybe because the oscillatory perfusion can provide a reciprocating flow to the cells.41 In addition, oscillatory fluid can provide sufficient nutrients and oxygen exchange and to remove metabolic waste in 3D porous scaffold during prolonged static culture in vitro. In this work, the number of dead cells on the scaffolds was relatively small during 3 weeks of culture time, possibly because oscillatory perfusion culture can rush out the dead cells from the scaffolds timely. Although stiffness-mediated expression of collagen gene expression42 and collagen protein expression43 has been noted previously on 2D platforms in vitro, the present results extended this observation by demonstrating enhanced collagen protein secretion of MSCs in 3D scaffolds with increasing stiffness. In addition, application of shear force on the cell-scaffold construct has been shown to increase the protein secretion and mineral deposition.44 Zhao and colleagues45 seeded the human MSCs on 3D poly (ethylene terephthalate) (PET) scaffold under static culture or perfusion culture conditions. After culture for 5 weeks, their results found the fluid shear stress can form an organized


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protein network.45 And in another study, Delaine-Smith et al.46 found that oscillatory perfusion could enhance the collagen secretion and apparent collagen organization of embryonic cell-derived mesenchymal progenitor cells. The present findings strengthen these previous results by the histology results and µ-CT data that oscillatory fluid perfusion could promote the collagen secretion and deposition of bone matrix. The data presented here demonstrated that the ECM deposition could be augmented on optimal matrix stiffness. The matrix stiffness not only can induce the osteogenic differentiation of MSCs, but also can affect the myofibroblastic activity of MSCs in the microfibrous scaffolds.47 In addition to regulation the stiffness of mixture matrix, the collagen and HA also have been applied in fabricating different bone scaffolds to enhance the bone defect repair. For instance, the HA was added to the silicon48 or poly(D, L-lactide-co-glycolide) (PLGA)49 to make 3D bone scaffolds by rapid prototyping technique. A recent study also reported that low-temperature additive manufacturing could print biomimic 3D bone scaffolds using collagen and HA composite material.50 These collagen or HA-based 3D scaffolds had good cytocompatibility, osteoconductivity, and easily modified for enhancing some defined characteristics and can effectively improve the osteogenic differentiation of MSCs in vitro and promote the bone regeneration in vivo. Fluid shear stress has the ability to control the fate of stem cells.51,52 Compared with the static culture, the MSC-scaffold constructs in the 3D perfusion bioreactor exhibited significantly higher calcium content, alkaline phosphatase activity, and OPN secretion.3 And in another study, de Peppo et al.53 seeded the human-induced pluripotent stem cells



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on bone scaffolds and observed the differentiation patterns of cells under perfusion culture. Their study revealed that the fluid shear stress could increase the ECM deposition and stimulate the expression of osteoblast-associated proteins. In this study, optimal Col/HA coatings on the decellularized bone presented higher osteogenic differentiation of MSCs under oscillatory perfusion culture than pure decellularized bone or other coated groups. This result may be explained by the mechanical properties of Col/HA coatings which initiate and direct stem cell lineage specification. In the study of Yeatts et al.,54 human MSCs cultured on nanofibrous electrospun scaffolds for 10 days in a tubular perfusion system bioreactor, then the constructs were implanted into rat femoral condyle defects. Three and six weeks later, defects implanted with tubular perfusion system cultured scaffold had the highest new bone formation than the statically cultured and acellular scaffolds. Our findings agreed with these previous results using oscillatory fluid perfusion could promote the expression of OPN and OC of MSCs on different 3D scaffolds. Furthermore, the MSCs on scaffold with optimal matrix stiffness have the highest expression levels of osteogenic markers under oscillatory perfusion culture. When developing a successful cell-based bone repair treatment strategy, it is essential to consider contributions from implanted cell population to bone regeneration after culture in different in vitro conditions. In the present study, seeded cell aggregates distributed uniformly in the whole 3D scaffold under oscillatory perfusion culture. Meanwhile, oscillatory fluid improved the survival rate of the cells while simultaneously reduced the dead cells in these engineering scaffolds. In addition, fluid shear force and


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matrix mechanics gave the nature of in vivo mechanical microenvironment on stem cells to enhance the bone formation when transplanted into bone defects. Moreover, in order to further improve the osteogenic ability of MSC-scaffold construct under the oscillatory perfusion culture, the osteogenic factors could add to the perfusion medium.

5. CONCLUSIONS In summary, the present study has proved that oscillatory shear stress not only helps to maintain high cell survival rate and uniform cell distribution throughout the scaffold after a relatively long period culture in vitro, but also facilitates osteogenic differentiation of MSCs. Furthermore, the findings also suggest that the oscillatory perfusion and matrix stiffness synergistically affect the osteogensis of MSCs within an appropriate range of parameters. The 3D scaffolds with optimal matrix stiffness in oscillatory perfusion bioreactors are a promising method for bone tissue engineering applications.

ASSOCIATED CONTENT

Supporting Information Additional details are available, including boundary and initial conditions for the mathematical model, schematic diagram of the perfusion system, and validation of the theoretical model. These materials are available free of charge via the Internet at http://pubs.acs.org.



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AUTHOR INFORMATION # These authors contributed equally to this work.

Corresponding Author *Yonggang

Lv,

Tel:

86-23-65102507,

Fax:

86-23-65102507,

E-mail

address:

[email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Guobao Chen# and Rui Xu# contributed equally to this work.

Conflict of Interest The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGMENT This work was supported in part by grants from the National Natural Science Foundation of China (11672051), Project Funded by China Postdoctoral Science Foundation (2015M582521), and the Fundamental Research Funds for the Central Universities (106112016CDJCR231214).

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Table of Contents/Abstract Graphic


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Responses of MSCs to 3D Scaffold Matrix ... - ACS Publications

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