Aging and MSCs
Ageing is associated with decreased fracture repair and reduced skeletal bone mass as a consequence of a net reduction in bone formation. This can result in osteoporosis with devastating socioeconomic consequences. As indicated, bone formation depends on MSCs present within bone marrow which in vitro give rise to CFU-F that differentiate into the osteogenic, adipogenic, fibroblastic, and reticular cell lineages (8,16,17,39). Studies on human osteoprogenitor number and age have been limited and contradictory. Nishida et al. (77) found that the ability to form alkaline phosphatase-positive (AP+) CFU-F was significantly reduced between the ages of 10 and 20 yr and then only gradually reduced after the age of 20. Similarly, D’Ippolito et al. (78) found a significant decrease with age in AP+-CFU-F in bone progenitors isolated from human vertebrae. Other workers have recorded decreases with age in CFU-F colonies or AP+-CFU-F in human (79), rat (80), and mouse (81) marrow. However, Oreffo et al. (82,83) in a study of 99 patients who were osteoarthritic, osteoporotic, or without evidence of metabolic bone disease, found no differences in CFU-F or AP+CFU-F with age, disease state, or gender, although a significant decrease in CFU-F colony size was observed. The decreased CFU-F colony size may be owing to replicative senescence (growth arrest) caused by reduced telomere length associated with age. Telomeres are repetitive DNA sequences that protect the end of chromosome during replication (84). During replication telomeres become shorter, eventually leading to chromosome instability resulting in replicative senescence (85). This acts as a countdown mechanism to limiting the proliferative capacity of cells. Telomere length decreases with age and hence could explain the limited proliferative capacity of mesenchymal cells in older patients (86). However, this process can be reversed by the overexpression of telomerase in MSCs by genetic manipulation (87–89). Overexpression of telomerase restores telomere length extending the replicative capacity of the cells indefinitely with the cells retaining their ability to differentiate into osteocytes, chondrocytes, and adipocytes. However, researchers are still uncertain for the safety of such immortalized stem cells. For example, in vitro, Serakinci et al. (90) have demonstrated that some populations of immortalized human MSCs lack contact inhibition, anchorage dependence, and also formed tumors in mice. However, others have demonstrated that when transplanted into mice, such cells were able to form bone with no evidence of tumor formation (88,89). These results suggest that the limited proliferative capacity of MSCs can be overcome and it may be a useful strategy in bone regeneration and repair but that caution must be taken to better understand any potential for neoplastic change.
Stenderup et al. (91) found the number and proliferative capacity of osteogenic stem cells was maintained during aging and in patients with osteoporosis. The bone loss associated with aging may reflect altered proliferative capacity of progenitor cells or altered responsiveness of CFU-F to systemic or locally released growth factors (92,93), leading to alteration in subsequent differentiation. The complex picture presented from these studies and the discrepancies therein may be attributable to different sample population sizes, populations selected, and laboratory protocols used, but indicate substantial modulation of MSCs with ageing.
Stem Cells, Genes, and Tissue
Engineering: Restoring Aging Bones Osteoporosis is currently defined as a systemic skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (94). Clinically, osteoporosis is recognized by the occurrence of characteristic lowtrauma fractures, which typically arise at the hip, spine, and distal forearm. It is estimated that around 40% of US white women and 13% of US white men of 50 yr of age will experience at least one clinically apparent fragility fracture at these sites during their lifetimes (95). However, taking into account sites other than the hip, spine, and distal forearm, the lifetime risk among women aged 50 yr might be as high as 70% (96). The medical costs of osteoporosis and its attendant fractures have been estimated in the US to be $ (17.9 × 109)/yr, with hip fractures accounting for one-third of this total. In England and Wales, hip-fracture patients alone take up 20% of orthopedic beds, with an estimated cost for all osteoporotic fractures of £ (1 × 109)/yr (97). Furthermore, the overall burden on the public health is set to increase dramatically over the next 60 yr because of the steep predicted increase in the proportion of elderly people in the population. Thus, worldwide, there were an estimated 1.66 × 106 hip fractures in 1990, a figure which is predicted to increase to 6.26 × 106 in 2050 if adequate preventative measures are not taken (96). Around 30–50% of the hip operations will require subsequent revision surgery and, in a significant proportion, bone augmentation will be necessary. With an increasing ageing population, overall health costs are set to rise. In addition, the observation that artificial prostheses, which are subjected to wear owing to lack of integration resulting in aseptic loosening, ultimately fail (reviewed in ref. 98), has further driven research activity to exploit the potential of MSCs in bone repair and regeneration (13,16,99). At present, regimes that encourage bone formation or delivery strategies for osteotropic agents such as the BMPs, which hold the promise of significantly increasing bone density, have proved elusive. Tissue engineering seeks to resolve these issues through a combination of stem or progenitor cells with appropriate growth factors and tailored three-dimensional scaffolds. Thus tissue engineering has been defined as the application of scientific principles to the design, construction, modification, and growth of living tissues (100). As a source of progenitor cells, it has long been known that bone has a vast capacity for regeneration from cells with stem cell characteristics. Kadiyala and co-workers have shown that culture-expanded bone marrow cells will heal a segmental bone defect following reimplantation (101).
Several groups have shown that MSCs and osteoprogenitor populations from a variety of species, including human MSCs, do give rise to osteogenic tissue within diffusion chambers (102–104). As detailed above, human bone marrow osteoprogenitors can be isolated and enriched using selective markers, such as STRO-1, from a CD34+ fraction (105,106) and readily expanded, indicating their potential for marrow repopulation (16,26,107,108).Adesirable extension in the use of MSCs for tissue regeneration would be the potential for the use of allogeneic populations allowing the deployment of cells from one or more donors, their preparation, and cryopreservation until required. Studies by Bartholomew et al. (109) and Di Nicola et al. (110) suggest that MSCs may be immune-privileged cells failing to elicit an immune response when combined with allogeneic lymphocytic cells. In support of such an approach, Arinzeh and coworkers (111) have shown that, at least at 4 and 8 wk, allogeneic MSCs aid regeneration in a critical-sized canine segmental defect. No systemic alloantibody production was detected over time (although a few allogeneic cells could be detected at 8 wk); these results add further support to the potential of allogeneic MSCs in cartilage and bone repair. Clinical data from Horwitz and colleagues (112,113) using human bone marrow-derived osteoprogenitors transplanted into children with osteogenesis imperfecta suggest some therapeutic effect of such an approach. Donor-derived MSCs (approx 2%) were capable of homing to the bone marrow and differentiating into the osteoblasts. In follow-up studies, the same group reported an increase in bone mineral content compared with age-matched controls, although the precise contribution of the donor cells to the clinical improvements recorded remains to be determined (114). Connolly (115) as well as Quarto and co-workers (116) have indicated the efficacy of autologous bone marrow stromal cells in the treatment of large bone defects. However, true engraftment of human MSCs, long-term biological effects on the stem cells at the implant site, as well as issues of cell plasticity remains to be defined.
MSC or osteoprogenitor delivery, whether by injection or with a matrix, is attractive and carries a reduced risk of morbidity. However, for large skeletal defects these cells will need an appropriate, designed, or “smart” vehicle/scaffold/matrix tailored to the shape and size of the defect and one which will provide mechanical competence. To aid this process, advances in scaffold technology and gene delivery offer the possibility of genetic modification of isolated and expanded cells in constructs to produce populations of progenitor cells overexpressing selected signaling molecules for bone regeneration (13,114,117–119). Lieberman and co-workers have shown regional cell and gene therapy using BMP-2-producing bone marrow cells on the repair of segmental bone defects in rats (120). Similarly, Breitbart and colleagues (121) have cultured periosteal cells retrovirally transduced with BMP-7 in a poly(glycolic acid) (PGA) scaffold in a critical sized calvarial defect model in rabbits. Several studies indicate human bone marrow stromal cells expressing BMP-2 by adenoviral infection may prove efficacious in bone regeneration (29,122–124) (Fig. 2). Musgrave and co-workers (125) reported on the use of direct adenovirus-mediated gene therapy to deliver active BMP-2 and produce bone in skeletal muscle, thus circumventing problems associated with delivery of BMP-2 and the requirements for cell expansion. However, limitations of associated immunogenicity in vivo, fate of adenoviral cells, longterm safety, and requirement for cell expansion in culture before viral infection and reimplantation remain significant hurdles before clinical use.
A variety of materials have been used for bone regeneration together with MSCs and osteoprogenitors, these include ceramics or materials based on hydroxyapatite, ceramic forms of β-tricalcium phosphate and composites of both hydroxyapatite and β-tricalcium phosphate (119). In recent years workers have sought to exploit biological cues that are necessary to mimic the cell–bone matrix interactions with the generation of biomimetic scaffolds based on poly(lactic acid), poly (-lactic-co-glycolic acid), as well as PGA (126,127). Healy and co-workers (128) and Dee and co-workers (129) have reported on the ability to modulate the adhesion of osteoblasts selectively and significantly by immobilized –Arg–Gly–Asp– (–RGD–) and –Phe–His–Arg–Arg–Ile–Lys–Ala– (–FHRRIKA), and Lys–Arg–Ser–Arg peptides (KRSR). In addition, Yang et al. (130) have shown the potential to promote human osteoprogenitor differentiation on RGD-coupled biodegradable scaffolds. Continued advances in gene delivery combined with development of smart designer scaffolds and materials in combination with selection of MSC and osteoprogenitor populations are needed to deliver on the promise of skeletal tissue engineering using MSC populations.