Adult bone tissue and periosteum can be used as sources of primary osteogenic cells 25 26 27 28 . Isolation techniques usually involve preparation of explant cultures from the dissected tissues, or enzymatic release of progenitor cells from the endosteal and periosteal layers (Figure 2a). Stepwise collagenase digestion has been used for the preparation of osteoblast-like populations with lower proportions of adherent stromal cells 25 29 . In some studies, fetal bone tissue has been used as a potential alogeneic source due to fast cell proliferation and has demonstrated osteogenic potential 30 .

In vitro, between 20 and 40 population doublings have been reported for primary bone and periostal cells 31 32 . Studies have indicated differences in proliferation rates of the bone cells isolated by different methods and originating from different donors, as well as age-related declines in cell proliferation 26 27 30 . Differences in the proliferation potentials of bone cells isolated from different sites have been observed 29 33 , similar to bone marrow stromal cells originating from different sites 34 .

In most studies, the expression of osteogenic markers (for example, increased alkaline phosphatase activity, synthesis of osteopontin, bone sialoprotein, osteocalcin and extracellular matix calcification) has been noted in the presence of the osteogenic supplements 1,25-dihydroxy vitamin D3, dexamethasone, β-glycerophosphate and L-ascorbic acid 35 36 , but only a limited amount of work has directly compared the functional potentials of osteogenic cells isolated from different sources 37 . In addition to osteogenesis, it has been reported that periosteal and endosteal populations also exhibit chondrogenic and adipogenic differentiation potential 31 32 .

Primary human bone and periosteal cells cultured on porous scaffolds formed bone-like tissue 11 38 39 . In separate studies, bone constructs have been engineered from periosteal cells and used clinically to enhance healing of periodontal defects (Table 1).

Taken together, these studies demonstrate that primary osteogenic cells can be isolated from tissues discarded during surgical procedures and used for in vitro studies 33 , and suggest that harvests of small tissue volumes from relatively accessible sites (for example, jaw bones during dental implant placement) could potentially be used for cell isolation and preparation of autologous grafts up to several millimeters in diameter and length. In contrast, due to donor site morbidity 40 41 42 and limited proliferation of primary cells, it would be difficult to envision routine preparation of large autologous grafts (several centimeters in diameter and length) from primary bone or periosteum-derived cells (Table 1). The applicability of such approaches will strongly depend on developing robust cell preparation procedures from source tissues that are inherently variable due to donor age, gender, health status, systemic conditions and genetic background.

Adult mesenchymal stem cells capable of differentiation into bone, cartilage, adipose, muscle, tendon, ligament and marrow stroma have been found in a variety of tissues, including bone marrow, adipose tissue, synovium, dental pulp, cord blood, umbilical cord and others 43 44 45 46 . For bone regeneration, the most studied source has been the bone marrow, as it was recognized early that its stroma contains bone marrow mesenchymal stem cells (BMSCs) capable of forming bone and cartilage 47 . Bone marrow transplantation is also being used clinically in combination with osteoconductive materials to augment bone healing 6 48 .

BMSCs are commonly isolated based on their adherence and growth on tissue culture plastics (Figure 2b). Alternatively, pure bone marrow aspirates can be used to immunoselect BMSCs using specific surface markers. The small initial numbers of immunoselected cells are then expanded in culture to obtain sufficient cell mass for therapeutic purposes. The number of stem cells (0.001 to 0.01% of the nucleated marrow cells) 43 varies between different patients and reportedly declines with patient age 46 . Additionally, marrow aspiration volume (up to 150 ml) and technique can influence the number of isolated stem cells 48 . BMSCs can, however, be culture-expanded to large numbers and have been reported to reach up to 50 population doublings in vitro 49 . Importantly, studies suggest that the osteogenic potential of BMSCs is maintained in older individuals 46 , and appropriate conditions in vitro (for example, culture on collagen substrate, growth factor supplementation of culture media) 50 51 can help maintain cell differentiation potential.

Adipose tissue stem cells (ASCs), discovered more recently 44 , represent another attractive source for bone tissue engineering due to their accessibility and potential for differentiation into osteogenic, chondrogenic, adipogenic and endothelial lineages 52 . Lipoaspirate volumes can range from 100 ml to several liters and contain a relatively high frequency of ASCs (1 to 5% of isolated nucleated cells) 53 . Cell isolation protocols usually include density gradient centrifugation of the collagenase-digested tissue (lipoaspirate or minced adipose tissue) and culture of the adherent cell population (Figure 2c). Similar to BMSCs, the numbers of isolated stem cells are influenced by the tissue harvesting procedure, as well as the site of tissue harvesting (for example, arm, thigh, abdomen, breast) 52 .

Several groups have reported the formation of bonelike constructs from BMSCs and ASCs cultured on porous scaffolds 54 55 56 , and noted positive effects of dynamic bioreactor culture on cell distribution and matrix formation 12 20 21 . Recently, our group has reported engineering of fully viable, clinically sized, and precisely shaped temporomandibular joint grafts by culturing BMSCs on anatomically shaped scaffolds in specially designed 'anatomical' bioreactors 57 . This study illustrates the feasibility of using adult stem cells for engineering functional human bone grafts, and underlines the importance of perfusion culture to support physiologic cell densities, as well as formation of dense, homogenously distributed bone matrix.

Survival of large engineered grafts after implantation remains an open question due to the need for immediate connection to the host vasculature, which is an unsolved problem of all tissue engineering. Various strategies for pre-vascularization are currently under investigation 7 . For example, Scherberich and colleagues 58 obtained bone constructs with intrinsic vascularization potential by culturing isolated adipose stromal vascular fractions on porous scaffolds in perfusion for 5 days. These efforts could enhance the survival of implanted grafts once the methods become available for the connection of the graft to the blood supply of the host.

A few clinical studies have reported on bone constructs prepared from adult stem cells and implanted to enhance bone regeneration. Importantly, adverse side effects of the transplanted cells have not been reported, and the authors suggested possible positive effects of transplanted cells on bone regeneration (Table 1).

Differentiation potentials of adult stem cells obtained from various sources are under investigation, as are the culture conditions required to achieve the functional properties of terminally differentiated cells. Another focus is determining correlations between the phenotypes of cultured cells and their potential for functional differentiation. Stem cells isolated from various tissues are frequently evaluated for the expression of surface antigens by flow cytometry 43 45 , and share common patterns between various BMSC preparations, and a highly conserved profile between ASC preparations 52 . In spite of the relative uniformity of marker expression, the cell potential to deposit bone matrix can vary quite significantly between different donors and cell populations 21 59 .

Simple models for evaluating the differentiation potential of cells are provided by micromass and pellet cultures. Pellets are prepared by centrifugation of several hundred thousand cells, and incubated in differentiation media for specific differentiation paths - in most cases osteogenic, chondrogenic and adipogenic. Micromass cultures are prepared by plating droplets of high cell density suspensions on tissue culture plates, which are also incubated in specific cell differentiation media. In both systems, high cell density helps mimic cell interactions and cell condensation events present during native development of cartilage and bone.

Osteogenesis and chondrogenesis can be measured quantitatively using molecular, biochemical and histological assays 12 21 . Bone formation capacity can also be evaluated in vivo - for example, in ectopic bone formation models 60 . In a recent study, correlations between bone marker gene expression and functional osteogenesis assays have been made to construct a mathematical model for predicting the bone forming capacity of the synovial and periosteal stem cells 60 . In future, such models could possibly be implemented in culture protocols to help develop robust procedures for manufacturing bone grafts.

Several reports of long-term BMSC and ASC cultures (≥ 4 months, ≥ 30 doublings) have indicated changes in cell cycle kinetics and the possibility of abnormal karyotype development, leading to malignant cell transformation 61 62 . These studies identified some limitations of ex vivo manipulation, which should be taken into consideration and explored further to ensure the biosafety of adult stem cells before their clinical application.

Pluripotent human embryonic stem cells (ESCs) can form any tissue of the body and have exhibited an unsurpassed (possibly unlimited) potential for proliferation in vitro 63 . ESCs were first successfully isolated and cultured in 1998 by Thomson and colleagues 64 , and have enormous value as a potential source of cells for regenerative medicine, as well as a model of early human development. In bone tissue engineering, ESCs could be used as a single source for the derivation of multiple lineages present in adult bone, including osteogenic cells, vascular cells, osteoclasts, nerve cells and others.

Compared to adult stem cells, ESCs require complex culture conditions: they are commonly derived from blastocyst-stage embryos and cultured on mitotically inactivated murine feeder cells in media supplemented with basic fibroblast growth factor and other factors 63 . ESCs grow in colonies, and are passaged as small aggregates by mechanical or enzymatic dissociation from the feeder cells. In recent years, progress has been made, and completely defined feeder-free conditions have been reported 65 . In an alternative approach, human feeder cells (for example, skin fibroblasts) have been used for ESC culture 66 .

Similar to adult stem cells, pluripotent ESCs are characterized by the expression of specific surface antigens, including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigens TRA-1-60 and TRA-1-81, and the absence of SSEA-1 67 . Other markers associated with undifferentiated ESCs are high alkaline phosphatase and telomerase activities, and expression of transcription factors Oct4, Sox2 and Nanog, which are crucial for the maintenance of pluripotency 68 . A standard test for confirming human ESC differentiation potential in vivo is the formation of teratomas after cell injection in immunodeficient mice. Pluripotency can also be evaluated in vitro by inducing differentiation in embryoid bodies (aggregates of cells cultured in suspension) and observing formation of tissues from all three germ layers 67 .

Spontaneous development of abnormal karyotypes and other genetic alterations have been observed during prolonged cultivation of ESCs 69 70 . Therefore, frequent monitoring of the karyotype is recommended, and further studies are needed to ensure stability and safety of the ESC-derived progenitor populations before their potential use in regenerative medicine.

Recently, the prospect of using ESCs for autologous therapies has gained attention with reports of induced pluripotent stem cells derived from adult differentiated cells 71 . Induced pluripotent stem cells share many characteristics with ESCs, including morphology, proliferation, surface antigens, gene expression, epigenetic status and pluripotency. Development of safer alternatives for cell reprogramming (for example, excluding genetic manipulation) could potentially provide a cell source for autologous therapy 72 . Currently, however, these cells present a unique opportunity to study the development and progression of genetic diseases in vitro.

The pluripotent nature of ESCs presents a challenge to the development of efficient protocols for directing cells into specific lineages. The embryoid body step has been an integral part of many differentiation protocols, including osteogenic differentiation. Cells capable of osteogenesis have been found in mixed populations of progenitor cells present in embryoid bodies after 4 to 5 days of culture 73 74 , and populations arising from co-cultures with primary bone and periodontal ligament cells 75 76 . A direct differentiation protocol excluding the embroid body step has also been tested, and seemed to enhance ESC osteogenesis in vitro 77 .

Alternatively, induction and isolation of mesenchymal stem cells (MSCs) from ESCs has been attempted, and these MSC-like progenitors have been subsequently directed into the osteogenic lineage (mostly in monolayer culture). In one study, MSC-like progenitors have been obtained by mechanical isolation of spontaneously differentiated cells from ESC cultures, followed by longer culture in confluent monolayers 78 . In another study, co-culture with stromal cells was used to induce differentiation, followed by immunoselection of a MSC-like population 79 . More recently, exposure of ESCs to serum and growth factor supplemented media 80 81 has been used to induce differentiation, and MSC-like progenitors have been expanded in a subsequent monolayer culture. Whereas these studies elucidate some of the factors involved in osteogenesis of ESCs, further work is needed to gain a better understanding of the developmental processes involved in specification to bone-forming cells, as well as to evaluate the stability and functionality of the obtained populations 82 .

In vivo implantation of stem cells genetically engineered to carry osteogenic genes has been shown to induce rapid bone formation, indicating the possibility of enhancing regenerative processes by combining cell and gene therapy strategies. It has been hypothesized that genetically modified cells exert both autocrine and paracrine effects, recruiting host cells to the site of implantation and resulting in enhanced osteogenesis 83 . Importantly, recruitment of host cells could allow for a reduced number of exogenous cells that need to be implanted. In many studies, adult stem cells have been engineered to express genes of the bone morphogenetic protein family (for example, BMP2, BMP4, and BMP7). Other genes of interest include those encoding transcription factors essential for osteoblast differentiation (for example, core binding factor α1 (Cbfa1), and Osterix), factors enhancing angiogenesis (for example, vascular endothelial growth factor), and bone formation antagonists (for example, noggin) for an additional level of control over bone formation, and combinations of several factors 83 . The challenges lay in efficiently delivering therapeutic genes into the cells without adenoviral and retroviral vectors - for example, by nucleofection (a form of electropermeabilization) - in order to increase safety and allow for the subsequent development of clinical applications.