Skeletal Stem Cells - Engineered Bone: Tissue Engineering Intelligence Unit - page 15

tendocytes. 3,64 Yet another study used whole bone marrow, rather than purified SSCs, to ini-
tiate a skeletal muscle differentiation. 18 Contribution of hematopoietic stem cells or other
potential stem cells present in marrow to these lineages should be excluded before giving all
credit to SSCs.
SSC in Vivo Transplantation as the Assay of Their Multi-Potentiality
In vivo transplantation has been a major tool for studying differentiation potential of stem
cells in general, and remains the “gold standard” for studying SSC multi-potentiality. Being
highly adhesive in nature, SSCs are very sensitive to the microenvironment in which they are
transplanted. Usually, no hard tissue is formed when an SSC suspension is injected subcutane-
ously or intramuscularly, when SSCs are implanted as a cell pellet without vehicle 33,63 or within
rapidly resorbed vehicles. 25,25 For chondrogenic differentiation, high cell density and relatively
anaerobic conditions are required, such as in micromass or pellet cultures. To initiate osteo-
genic differentiation, SSCs require the presence of a scaffold of a particular composition and
architecture, depending on the animal species, that can support their growth and differentia-
tion. Based on vehicle design, SSC transplantation techniques can be divided into two groups:
closed and open.
In a closed system, cells are transplanted within diffusion chambers constructed of Millipore
filters (0.22 µ m or 0.45 µ m porosity), such that the transplants receive nutrients but have no
direct contact with the host cells. SSCs transplanted in closed systems form bone, cartilage,
fibrous tissue, and sometimes fat. Bone is developed adjacent to the filters, while cartilage is
found towards the center of the chamber 1,2 suggesting that SSC differentiation may be affected
by the nutritional environment, including oxygen gradients. This conclusion is supported by
the observation that in chambers with a narrow gap between the filters (0.1 mm), bone with-
out cartilage is formed, while in chambers with a wider gap (2 mm), both bone and cartilage
are developed. 23
Transplantation into an open system (usually subcutaneously or under the kidney capsule)
allows contact with recipient cells and is followed by rapid vascularization of the graft. In these
conditions, SSCs rarely differentiate into cartilage, but in addition to bone and fibrous tissue,
they form hematopoiesis-supporting reticular stroma and associated adipocytes (Fig. 1A). Vast
fields of hematopoiesis are developed in close proximity to the new bone. In the absence of
bone formation, hematopoiesis is never observed. In open transplants, SSCs demonstrate
species-specific sensitivity to the nature of transplantation vehicle. Mouse SSCs form bone
when transplanted within collagen sponges. 25,44 Human SSCs, however, form very little bone
or no bone at all in vehicles such as collagen sponges, human demineralized bone matrix,
polyvinyl sponges, and poly(L-lactic acid). 45,58 Human SSCs consistently formed extensive
bone only in vehicles containing hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic in
the form of blocks, powder, or Collagraft TM (mixture of ceramic powder with bovine collagen
type I). 43,43 Furthermore, the development of the new bone is strongly influenced by size and
shape of vehicle particles 46 emphasizing, once again, the importance of vehicle structure for
SSC osteogenic differentiation.
In addition to studying SSC populations en masse, in vivo transplantation assay allows
analysis of the heterogeneity of SSC populations. For this purpose, progeny of individual CFU-Fs
can be expanded and transplanted separately. This type of assay provided similar results for
SSCs derived from several species. Mouse and guinea pig SSC colonies were transplanted in
vivo and in both cases, approximately 20% of the colonies formed bone with or without he-
matopoiesis, while the remaining colonies formed just fibrous tissue or no donor tissue at all. 12
Rabbit single-colony derived SSC strains were transplanted in diffusion chambers, either
autologously or into immunodeficient mice, and bone, either with or without cartilage, was
formed by 48.3% 23,31 and by 36.8% of the strains. 4 Human single-colony derived SSC strains
were transplanted into immunodeficient mice in HA/TCP powder and Collagraft. Out of 34
strains, 20 (58.8%) formed bone while the remaining strains formed fibrous tissue. Among the
20 osteogenic strains, 8 (23.5%) developed extensive bone accompanied by hematopoiesis,