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

*Clinical Implementation of Bone Regeneration and Maintenance*

achieve a sterility assurance level (SAL) of 10<sup>−</sup><sup>6</sup>

fetal bovine serum, 100 U mL<sup>−</sup><sup>1</sup>

The demineralized fibers were then freeze-dried, placed in final packaging, and treated via low-dose, low-temperature gamma irradiation, at a level necessary to

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

penicillin, 100 μg mL<sup>−</sup><sup>1</sup>

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-

**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

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

**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

streptomycin, and 2 mM

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

5:2009, percent viability less than 70% indicates a cytotoxic effect.

was used to determine differences in metabolic activity over time.

.

**36**

cm2

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 in ng protein/g of demineralized fibers.
