Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Summary

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Abstract

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

Protocol

1. Cell Isolation & Expansion

1. All patient consent and experimental protocols were reviewed and approved by Stanford University Institutional Review Board (Protocol #2188 and #9999).
2. Obtain human subcutaneous adipose tissue from elective lipoaspiration procedures under local/general anesthesia.
3. There will be two layers in the lipoaspirate (Figure 1A). The supernatant contains the vast majority of the processed cellular material. The bottom layer is mostly injected saline. Adipose-derived stromal cells can be harvested from either layer, but the yield is much greater from the supernatant.
4. To iso....

Discussion

Since the isolation of adipose-derived stromal cells² from lipoaspirate, these cells have been differentiated into a wide variety of cellular lineages. Adipose tissue is from mesodermal origins and therefore, multipotent adipose-derived stromal cells will likely be most effective with application towards a mesodermal lineage. The ability to generate skeletal tissue is especially critical given the shortage of donor sites for an autograft and the inherent limitations of synthetic material including infection, r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipoaspirate Harvest
PBS	Gibco	10010-023
Hank's Balanced Salt Solution	Cellgro	21-023-CV
Collagenase	Sigma	C6885-500MG
Cell Strainer 100 μm	BD Falcon	352360
Steri-top 500 ml .22 μm filter	Millipore	SCGPT05RE
Calvarial Defect
Z500 Brushless MicromotorsUM50C	NSK	NSKZ500
Circular Knife 4.0 mm	Xemax Surgical	CK40