CONFORM® with Q-PACK®: An Evaluation of a

CONFORM® with Q-PACK®:
An Evaluation of a Fully Demineralized
Scaffold with and without Q-PACK
Technology Seeded with Human
Mesenchymal Stem Cells
SUMMARY
The CONFORM SHEET® with Q-PACK is a fully demineralized cancellous scaffold packaged in a hydrated state. Its
intrinsic properties allow it to be compressible and wickable, as well as have the ability to be combined with bone
marrow aspirate (BMA), blood, or other fluids. Due to the inherent properties of the natural scaffold in combination
with the proprietary demineralization and pH restoration processes that are used, CONFORM SHEET aids in bone
formation by exposing bone morphogenetic proteins and providing a cell friendly environment. Clinically, bone
marrow aspiration is used to recover cells to add to the allografts. This technique provides cells and additional
biological growth factors that assist in the healing and regeneration of bone. Cells can populate the demineralized
cancellous scaffold through the use of BMA or migration of host cells into the scaffold following implantation.
To evaluate the cellular attachment and distribution characteristics, an in vitro study was conducted in which
demineralized cancellous scaffolds with and without the Q-PACK technology were seeded with human mesenchymal
stem cells (hMSCs). Following specified incubation periods, the tissue was assayed to determine cell counts, and
confocal microscopy was employed to evaluate cell distribution. The results demonstrate that both demineralized
cancellous scaffolds with and without the Q-PACK technology provide similar environments where cells can attach
and produce extracellular matrix.
MATERIALS AND METHODS
Scaffold Preparation: Cancellous bone from two donors was demineralized using a proprietary process that fully
demineralizes the tissue as well as restores the pH to neutral levels. Tissue from each donor was split into two
groups where one group was lyophilized and the other group was processed using the Q-PACK technology. The
tissue was not rinsed prior to preparation in order to represent the worst case sceneario for cell seeding with residual
ethanol still present. The tissue was subsequently cut into cylinders with a diameter of 4.0mm ± 0.5mm and a height
of 2.5mm ± 0.5mm.
Cell Seeding: The lyophilized and Q-PACK tissue was rehydrated and placed inside an agarose gel coated transwell
plate. Each sample was then seeded with 500,000 hMSCs cultured from commercially available hMSCs (Lonza).
The tissue was incubated at 37°C for specified time periods and assayed to determine cell counts and analyze cell
distribution.
Cell Count: Cell counts were obtained using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) at two
time points: 30 minutes, and 3 hours. The assay was performed in triplicate for each donor.
Cell Distribution: Prior to seeding onto the tissue, cells were fluorescently stained with CellTracker Green CMFDA
(5-chloromethylfluorescein diacetate) (Invitrogen), and the tissue was fluorescently stained with Alexa Fluor™
633 (Invitrogen). Once stained, the cells were seeded onto the tissue and incubated. At incubation periods of 30
minutes, 3 hours, and 24 hours, samples were fixed in 4% formalin and imaged using confocal microscopy.
RESULTS
Cell Count: The CellTiter-Glo Luminescent Cell Viability Assay quantifies the amount of adenosine-5’-triphosphate
(ATP) by lysing the hMSCs and producing a luminescent signal proportional to the amount of ATP. From the
luminescent signal, the cell number can then be calculated, as shown in Figure 1.
Cell retention is based on the method of cell
Number of Cells
Cell Number for Seeded Samples
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
application as well as the porosity of the scaffold.
The results showed that once seeded onto either
tissue, the cells remained viable and metabolically
active, as the cell counts for each sample remained
similar between the Q-PACK and the lyophilized
Sheets (p=0.7971). This demonstrated that Q-PACK
Lyophilized
Q-Pack
30 Minute
tissue provides an equally cell-friendly environment
as the lyophilized tissue.
3 Hour
Incubation Period
Cell Distribution: Confocal microscopy was used to
obtain images of the hMSCs seeded onto each of the
Figure 1: The average cell number for each sample group
seeded with hMSCs. The numbers are an average of two
donors ± the standard error.
scaffolds. Over an incubation period of 3 hours, 24
hours and 7 days the cells attached to the surface
of the tissue. By 24 hours, the cells have begun
to produce extracellular matrix, demonstrating
biocompatibility of both scaffolds.
Q-PACK
Lyophilized
30 minutes
3 hours
24 hours
Figure 2: Confocal images of hMSCs seeded onto fully demineralized cancellous scaffold that was lyophilized and
scaffolds that were packaged using Q-PACK technology. The cells were fluorescently stained green while the scaffold
was stained red.
CONCLUSION
These study results demonstrate that tissue packaged with the Q-PACK technology provides a biocompatible
environment for human mesenchymal stem cells that is equivalent to the lyophilized packaged tissue. The hMSCs
attach and produce extracellular matrix in both the test and control articles. After addition of hMSCs to each scaffold
the cell viability remains consistent for 3 hours and by 24 hours the cells have begun to produce an extensive
extracellular matrix.
Musculoskeletal Transplant Foundation
125 May Street,
Edison, NJ 08837, USA
All in vitro data on file at MTF.
In vitro test results may not necessarily be indicative of clinical performance.
®
CONFORM , CONFORM SHEET and Q-PACK are registered trademarks of the Musculoskeletal Transplant Foundation
CellTiter-Glo® is a registered trademark of Promega.
©2013 Musculoskeletal Transplant Foundation 7/13 00029 CI
®
®

Download Report