Abstract
3.2 Materials and Methods .1 Fabrication of Scaffolds
3.2.5 In vitro Biological Studies
3.2.5.1 Osteogenic Differentiation Potential Assessment Using hMSCs
The conditioned scaffolds were statically loaded with 3 x 105 hMSCs suspended in 30 μL of expansion media per scaffold and the cell seeded scaffolds were placed in incubator (37 °C in a humid atmosphere with 5 % CO2) for 4 hours to allow cell adhesion on the scaffolds after which the cell seeded constructs were flooded with hMSCs expansion media. After 24 hours post seeding the expansion media was replaced with osteogenic induction media (HiPer®
high glucose Dulbecco’s modified media supplemented with 10 % (v/v) foetal bovine serum, 10 mM β-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich) and 0.2 mM ascorbic acid (Sigma-Aldrich). The osteogenic media was changed every 48 hours till 14 days.
Cell Proliferation, Cell Cycle and Viability Assessment
For evaluating the cell proliferation, Alamar blue dye (Invitrogen, USA) reduction assay was used, following the manufacturer’s protocol at regular intervals. Briefly, the cell seeded scaffolds were incubated with 10 % (v/v) of the dye and incubated for 3 hours. After incubation, 100 μL of the culture media was read using a microplate reader (Tecan Infinite Pro, Switzerland) at 570/600 nm. The results are presented as the normalized value of the dye reduced, which is proportional to the number of viable cells present in the scaffolds. The viable cells within the scaffolds after 7 days were imaged using calcein-AM (Sigma-Aldrich). The cell seeded matrices were washed with PBS and incubated with PBS with 40 nM calcein-AM for 15 minutes. The dye solution was removed washed with PBS twice and cell seeded scaffolds were imaged using a fluorescent microscope (EVOS XL digital microscope, Thermo Fisher Scientific, USA) with live viable cells appearing green.
Biochemical Analysis Alkaline Phosphatase Assay
Alkaline phosphatase (ALP) activity was assessed to determine the osteogenic differentiation fate of the seeded hMSCs using an alkaline phosphatase assay kit (Abcam, UK) following the manufacturer’s protocol. At day-1, 7 and 14 the cell laden scaffolds were lysed using cell lysis buffer (20 mM Tris-HCl (Merck, India) (pH 7.5), 150 mM NaCl (Himedia, India), 5 mM MgCl2 (Himedia, India), and 0.5% Triton-X100 (Sigma-Aldrich) and the cell lysate was used for membrane bound ALP estimation. Similarly, the spent cell culture media was also used for soluble ALP estimation. The ALP activity obtained as U/ mL was normalized with the total DNA content for both the membrane bound and soluble ALP and represented as U/ μg DNA.
Total Collagen Estimation
To determine the amount of collagen secreted by the hMSCs osteogenically committed on the scaffolds was estimated following a previously published protocol [288], using sirius red based colorimetric assay with rat tail collagen (Sigma-Aldrich) (0−250 μg/mL) as standard.
Briefly, the cell laden scaffolds at day 1, 7 and 14 were digested in pepsin digestion buffer (0.1 M acetic acid, 0.5 M NaCl and 1 mg/mL pepsin (Sigma-Aldrich). 100 μL of the digesate from each sample was allowed to dry in 96 well plate at 37 °C overnight and treated with 100 μL of 1 mg/mL direct red 80 (Sigma-Aldrich) saturated with picric acid for 1 hour. The dye solution is removed washed with 0.01 N HCl and the samples are resolved using 100 μL 0.1 N NaOH and the absorbance were recorded using microplate reader at 550 nm. The amount of collagen determined is normalised with the amount of total DNA content and the data is presented as μg collagen/ μg DNA.
Western Blot Analysis
Immunoblots were performed to detect the expression of hypoxia inducible factor 1α (HIF-1α) on the protein level. Cell laden scaffolds were lysed using lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 % NP-40 (Sigma-Aldrich), 1 mM ethylene glycol-bis(2- aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich). The protein lysates were centrifuged and protein concentration was estimated using Bradford’s reagent (Sigma- Aldrich). 50 μg of total protein per sample were loaded per lane and resolved through SDS- polyacrylamide gel electrophoresis in 10 % separating gel. The resolved proteins were electroblotted on to poly(vinylidene fluoride) (PVDF) membranes (Sigma-Aldrich) and
blocked for 1 hour using 5 % (w/v) bovine serum albumin (Himedia, India) in tris buffered saline (TBST) (0.02 M Tris, 0.9 % (w/v) NaCl, 0.05 % (v/v) Tween-20 (Sigma-Aldrich). The membranes were washed with TBST and incubated with primary mouse monoclonal antibody against human HIF-1α (Abcam, UK; 1:200 dilutions) at 4 °C overnight. The expression of HIF- 1α was studied relative to GAPDH which was used as loading control (mouse monoclonal against human GAPDH, (Abcam, UK, 1:1000 dilutions). The blots were washed and incubated with horse radish peroxidase tagged secondary goat anti-mouse IgG (Abcam, UK, 1:10000 dilutions) for 1 hour. After a brief washing step, the blots were visualized using enhanced chemiluminescence method (ClarityTM Western ECL Kit, Bio-rad laboratories, USA) through a gel documentation system (Gel Doc XR+ system, Bio-rad laboratories, USA) and the densiometric analyses were carried out using Image-J software (NIH, USA).
Gene Expression Studies
For assessing the expression of osteogenic markers of the committed hMSCs, the relative gene expression of human runt-related transcription factor-2 (RUNX2), human bone sialoprotein (BSP) and human osteocalcin (OCN) was calculated with reference to the house keeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (listed in Table 3.2). Total RNA content was isolated using TRI reagent (Sigma-Aldrich). One microgram of RNA was reverse transcribed using high capacity reverse transcription kit (Applied Biosystems, Invitrogen, USA) in a thermal cycler machine (Applied Biosystems Veriti, Thermo Fisher Scientific, USA). The expression level of genes was quantified using Power SYBR PCR master mix (Applied Biosystems, Invitrogen, USA) in a real-time PCR machine (Applied Biosystems 7500, Thermo Fisher Scientific, USA).
Histological Assessment and Immunostaining
The cell laden scaffolds after 14 days were fixed using neutral buffered formalin (Sigma-Aldrich). The fixed constructs were then put through to ethanol-xylene dehydration- clarification procedure and embedded in paraffin wax (Merck, India) and further sectioned using a manual microtome (Leica Biosystems, Germany) to get 10 μm sections. The slices were stained with hematoxylin and eosin (Sigma-Aldrich, USA) to assess the cell distribution within the scaffolds. For immunostaining, the sections were blocked with 5 % BSA in PBS for 1 hour, followed by incubation by primary rabbit polyclonal antibody against osteopontin (OPN) (1:1000 dilutions) (Abcam, UK) for 1 hour and finally with FITC conjugated secondary goat anti-rabbit antibody IgG (Abcam, UK, 1:2000 dilutions) for 1 hour. The sections were counterstained with 1 μg/mL Hoechst-33342 (Sigma-Aldrich, USA) and mounted. The stained
sections were visualized using a fluorescent microscope (EVOS XL digital microscope) and represented images are presented.
3.2.5.2 Induction of Osteoclastogenesis and Investigation of Remodelling Effectiveness of