**2.2 Cytotoxicity testing of L-MDF using L929 mouse fibroblasts**

The cytotoxic potential of L-MDF were quantitatively evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using L929 mouse fibroblasts. Three samples of 2.5 cc from each of the six donors (*n* = 18) were rehydrated with 5 mL sterile saline (0.99% w/v sodium chloride in water). Sample extracts were prepared by incubating 0.2 g of each sample with 1 mL of extraction medium (Minimum Essential Medium supplemented with 10% v/v fetal bovine serum, 100 U mL<sup>−</sup><sup>1</sup> penicillin, 100 μg mL<sup>−</sup><sup>1</sup> streptomycin, and 2 mM l-glutamine) for 24 ± 2 h at 37 ± 1°C. Negative and positive controls were prepared similarly. Extraction medium alone was used as an untreated control "extract" for quantitative comparison of results. L929 mouse fibroblasts were cultured in 96-well microplates to half-confluency and subsequently exposed to 100 μL of sample or control extracts for 24–26 h at 37 ± 1°C. Following extract exposure, cell viability of each well was measured using a MTT assay. The average results for each group were normalized to the untreated control to determine a percent viability. Per ISO 10993- 5:2009, percent viability less than 70% indicates a cytotoxic effect.

#### **2.3** *In vitro* **metabolic activity of seeded bone marrow-mesenchymal stem cells**

Human bone marrow-derived mesenchymal stem cells (BM-MSCs) seeded on L-MDF were measured for metabolic activity using an alamarBlue® assay (Bio-Rad, Raleigh, NC) over the course of 7 days. L-MDF from six donors were placed in triplicate in low-attachment 24-well cell culture plates at a density of 13.1 mg of fiber per cm2 and seeded with BM-MSCs at 62,500 cells per well on day 0. BM-MSCs without fibers served as the control. After 2–4 h in culture, 1 mL of complete media was added to each well, followed by incubation at 37°C. Samples remained in the incubator until specific time points designated for analysis, at which point media was replaced. The metabolic activity of cells adhered to the fibers was measured after 1, 4 and 7 days in culture. At each time point media was aspirated and replaced with 1 mL of 10% alamarBlue reagent and incubated for an average of 2 h at 37°C. The solution was collected from each sample, centrifuged to pellet any debris, and measured in a 96-well plate at 544 nm excitation/592 nm emission. Fluorescence was recorded using relative fluorescence units (RFUs), and values were normalized to its time-matched control. A one-way ANOVA in conjunction with a Tukey post-hoc was used to determine differences in metabolic activity over time.

#### **2.4** *In vitro* **cellular attachment of seeded bone marrow-mesenchymal cells**

Scanning electron microscopy (SEM) was used to qualitatively evaluate the attachment and morphology of cells seeded on four L-MDF samples (25 ± 1 mg) at 0.5–1 h, 1 and 7 days in culture. The fibers were placed in separate glass scintillation vials with 1 mL of complete media and incubated at 37°C. Following incubation, excess media was aspirated and BM-MSCs were seeded at 100,000 per cells per vial. At each time point, corresponding vials were removed from the incubator, excess media was removed, and 3 mL of 2.5% glutaraldehyde in cacodylate buffer was added to fix the samples. Cell-seeded samples were rinsed in 0.1 M cacodylate buffer, incubated in 1% osmium tetroxide for 60 min, and then dehydrated in a series of ethanol solutions increasing in concentration up to 100%. Samples were then dried via evaporation of a chemical drying agent, hexamethyldisilazane (HMDS). Prior to imaging, all samples

**37**

**3. Results**

**3.1 Cytotoxicity**

*Cell Attachment and Osteoinductive Properties of Tissue Engineered, Demineralized Bone Fibers…*

were sputter coated in gold palladium for 200 s at 60 mA, then secured to a holder that was placed inside a vacuum-sealed imaging chamber. Samples were then imaged at a magnification 3000× using a Zeiss Gemini HD Scanning Electron Microscope.

L-MDF were analyzed for the presence of BMP-2 and BMP-7 using an enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis MN). L-MDF from six donors were weighed (30 ± 10 mg per donor) and placed in microcentrifuge tubes. Samples were then rehydrated with 5 μL of Dulbecco's Modified Eagle Medium (DMEM) per milligram of fiber, followed by the addition of purified collagenase (14.47 Units/mg of fiber). The samples were digested at 37°C with constant mixing for 16–18 h. Digestion solutions were centrifuged to remove remaining undigested components and the supernatants were collected for testing. The resulting solutions were analyzed for BMP content in triplicate using an ELISA assay. The measured BMP content was averaged across all six donors and results were reported

The osteoinductive potential of L-MDF was evaluated using an *in vivo* athymic

Cytotoxicity assay results showed that negative and positive controls behaved as expected (i.e., percent viability ≥70% for the negative control groups and <70% for the positive control groups). The average percent viability for negative and positive controls were 94 and 4%, respectively. The average percent viability of L-MDF (91%) was above the 70% threshold, and thus, based on the criteria of the protocol

Overall, the cellular activity of the BM-MSCs was shown to significantly increase

mouse model at NAMSA (Northwood, Ohio) following American Society for Testing and Materials (ASTM) F-2529 guidelines. Four 20–25 mg replicates of L-MDF were rehydrated with 100–150 μL of sterile 0.9% w/v sodium chloride and loaded into 0.3 cc sterile syringes. Samples were then compressed to remove excess solution, and implanted bi-laterally between the biceps femoris and superficial gluteal muscle of athymic mice. All mice were euthanized 5 weeks post-implantation by carbon dioxide inhalation. Explants were fixed with 10% formalin and bisected along the long axis. Bisects of each explant were paraffin embedded, and three slides were generated each with 4–6 μm-thick tissue sections. Once stained with hematoxylin and eosin (H&E), slides were evaluated by a blinded pathologist. The presence of cartilage, chondroblasts, chondrocytes, osteoblasts, osteocytes, osteoid, newly formed lamellar bone, and bone marrow were evaluated as new bone ele-

ments as they are indicators of endochondral bone formation process.

and ISO 10993-5 guidelines, L-MDF are considered to be non-cytotoxic.

**marrow-mesenchymal stem cells**

**3.2 L-MDF supports attachment and sustained metabolic activity of bone** 

over the course of the 7 day investigation. The results indicated that cells seeded on L-MDF showed a significant increase in proliferation between days 4 (51.3 ± 1.2 RFU) and 7 (59.5 ± 1.5 RFU) compared to day 1 (21.3 ± 0.8 RFU) (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.88290*

**2.5** *In vitro* **growth factor analysis**

in ng protein/g of demineralized fibers.

**2.6** *In vivo* **osteoinductive potential (OI)**

were sputter coated in gold palladium for 200 s at 60 mA, then secured to a holder that was placed inside a vacuum-sealed imaging chamber. Samples were then imaged at a magnification 3000× using a Zeiss Gemini HD Scanning Electron Microscope.
