**3.3 L-MDF contains important growth factors and demonstrates new bone formation** *in vivo*

The ELISA results indicated the presence of growth factors in L-MDF. The average BMP-2 and 7 concentrations in the samples fibers were 11.24 ± 1.49 and 85.78 ± 6.84 ng/g, respectively (**Figure 4**).

Additionally, in the athymic mouse muscle pouch model, histological analysis revealed new bone elements around and within the implanted scaffold at time of sacrifice (5 weeks; **Figure 5**). Panel A shows a set of merged images that illustrate

**Figure 4.**

*Clinical Implementation of Bone Regeneration and Maintenance*

SEM images confirmed BM-MSC attachment to L-MDF within 30 min of seeding. Cells appeared rounded with numerous folds and ridges and minimal surface contact (**Figure 3A**). After 1 h in culture, BM-MSCs became elongated and began spreading and increasing surface contact with the fibers (**Figure 3B**). After 1 day, imaging showed flattened cells with multiple adhesion points and cellular extensions as well as extracellular matrix (ECM) secretion (**Figure 3C**). By day 7 in culture, BM-MSCs infiltrated between fibers and demonstrated cell-to-cell interactions (**Figure 3D**).

*Representative SEM images illustrating the morphology of cells attached to L-MDF. Following culture for 30 min (A), 1 h (B), 1 day (C) or 7 days (D), respectively, the samples were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy. Images are representative of all samples evaluated and were taken at 3000× magnification. Scale bar represents 10 μm. Images were pseudo-colored in Adobe Photoshop to* 

*Proliferation of BM-MSCs attached to L-MDF over 7 days. The average relative fluorescence unit (RFU) values for each set of triplicate test samples were normalized to the average RFU of the corresponding control group (fibers of the respective donor cultured without cells) for all six donors. Asterisks represent statistically* 

**38**

**Figure 3.**

**Figure 2.**

*distinguish the cells (in yellow) from the fibers.*

*significant differences from day 1 proliferation activity.*

*BMP-2 and BMP-7 content in L-MDF. L-MDF produced from six different donors were digested in collagenase for 16–18 h. Using ELISAs, the resulting digestion solutions were tested for BMP-2 and BMP-7 content in triplicate (mean* ∓ *SE).*

#### **Figure 5.**

*H&E staining of explants from an athymic nude mouse implanted with L-MDF (\*). Merged set of H&E images showing new bone elements present in the entire explant at 35 days post-implantation (4× objective). Expanded areas show the presence of new bone elements such as cartilage (^), chondroblasts/cytes (#), bone marrow (\$), new blood vessels (&), and new bone (+) around L-MDF implant (\*) at 35 days.*

new bone elements present in the explant (4× objective). Panels B and C highlight the presence of new bone elements such as cartilage, chondroblasts/cytes, bone marrow, new blood vessels, and new bone.
