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Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.
This is described in details in the adjacent article "Purification of Perivascular Stem Cells from Human White Adipose tissue", by M. Corselli et al.
As both the calvarial and femoral defects are critical-sized, no significant healing should be expected without treatment with growth factors or exogenous stem cells.
In terms of surgical maneuvers, the muscle pouch dissection should be along fascial planes and thus minimal bleeding should be encountered. Even though the muscle pouch model is performed bilaterally, the mouse should be walking with ease on postoperative day 1. For the calvarial defect, bleeding is encountered but can be soaked with a Q-tip. Extreme care should be taken not to injure the underlying dura mater - as this will interfere with normal healing. For the FSD model, care is taken not to injure the major blood vessels so as not to cause excessive bleeding or the femoral nerve to prevent neurologic damage. Kirschner wires are drilled with gentle pressure so as not to damage the cortical bone in the process.
Figure 1. Preoperative preparation for Femoral Segmental Defect (FSD) in Athymic Rats. Male rats (12-14 weeks old) are anesthetized under isoflurane inhalation. The femur is scrubbed and prepped per standard protocol with betadine.
Figure 2. Surgical exposure for Femoral Segmental Defect (FSD) creation. A 27-30 mm longitudinal incision is made over the anterolateral aspect of the femur. The lateral aspect of the femoral shaft is then exposed by separating the vastus lateralis and biceps femoris muscles.
Figure 3. Fixation for Femoral Segmental Defect (FSD) creation. A polyethylene plate (length, 23 mm; width, 4 mm; height, 4 mm) is placed on the anterolateral surface of the femur. The plate contains six pre-drilled holes to accommodate 0.9 mm diameter threaded Kirschner wires. Taking the plate as a template, six threaded Kirschner wires are drilled through the plate and both cortices. Next, a 6 mm mid-diaphyseal defect is created. Once this is performed, a custom-made scaffold is directly inserted which exactly fits the defect site (not shown).
Figure 4. Example of Calvarial Defect Postoperative. A 4 mm, circular calvarial defect is created in the right parietal bone in athymic mice. Imaged here is a defect site implanted with PSCs at 8 weeks postoperative. Note the presence of new bone within the defect site.
The isolation of PSCs is well described elsewhere1-3, including a separately submitted JoVE publication specifically addressing PSC isolation protocols and methods. The specific purpose of this article is to describe and demonstrate 3 protocols for PSC in vivo application for bone formation/regeneration. The SCID mouse muscle pouch is a commonly described model for ectopic human bone formation7. Important differences exist between ectopic and orthotopic (defect) models for bone, including paracrine interaction with host bone-forming cells8 as well as an abundance of osteogenic signaling factors present in the skeletal defect microenvironment. Two defects are presented here, a calvarial defect8 and femoral segmental defect4. Both are well-documented to be critical sized (i.e. will not heal on their own).
Interesting differences exist between calvarial and femoral defects. First, the cell:cell interaction between xenografted PSCs and endogenous cells is very different. In terms of a calvarial defect, PSCs interact with the underlying dura mater (the outermost layer of the meninges), as well as those osteoblasts and periosteal cells circumscribing the defect site. Importantly, the interaction between implanted cells and surrounding osteoblasts8, or implanted cells and underlying dura (Levi et al., in press) are critical for normal stem cell mediated osteogenesis to proceed. In terms of the femoral segmental defect (FSD), xenografted PSCs are exposed to a very different cell and cytokine environment. For example, the FSD site is composed of the marrow and accompanying mesenchymal stem cells, as well as the endosteum, periosteum and long-bone osteoblasts. Theoretically, each cell has its own reaction to injury, and each may have cell:cell interactions with PSC xenografts.
Other clear differences exist between calvarial and femoral defects. The calvarial bones initially form through intramembranous ossification, while the long bones form through a cartilage intermediate (endochondral ossification). Moreover, the reparative process post-injury also mimics these developmental origins. Post-FSD, cartilage callus formation is observed, whereas no cartilage intermediate is formed within a parietal bone defect. Finally, the embryonic origin of the skull may differ from that of the long bones. The majority of the skull (including perivascular cells – pericytes – in the whole head region) is derived from the neural crest (mesectoderm), while the appendicular skeleton is of paraxial mesoderm derivation9. All these differences may result in significant differences in terms of PSC-mediated bone repair.
The use of PSCs has several benefits over traditional adipose-derived stromal cells (ASCs). PSCs do not require culture and are a purified cell population which does not include other stromal cells that do not participate in – and can even negatively regulate - osteogenic differentiation, such as endothelial cells10. In contrast, for example, clonal analyses of ASCs show that only a subpopulation are capable of undergoing osteogenic differentiation in vitro11. Ultimately, skeletal tissue engineering efforts will likely incorporate an osteocompetent stem cell (such as PSCs) with exogenous growth factors and an osteoconductive scaffold (such as HA-PLGA used in the present methods) so as to best heal skeletal defects.
K.T, B.P., and C.S. are inventors of perivascular stem cell-related patents filed from UCLA. Drs. K.T, and C.S. are founders of Scarless Laboratories Inc. which sublicenses perivascular stem cell-related patents from the UC Regents. Dr. Chia Soo is also an officer of Scarless Laboratories, Inc.
This work was supported by the CIRM Early Translational II Research Award TR2-01821, NIH/NIDCR (grants R21 DE0177711 and RO1 DE01607), UC Discovery Grant 07-10677, AWJ and RKS have T32 training fellowships awards (5T32DE007296-14), JNZ has a CIRM training fellowship (TG2-01169).