Osteoblasts that provide bone growth and repair are located in the inner layer of the

Osteoprogenitor cells

Osteoprogenitor cells, which originate from MSCs and differentiate into osteoblasts. They also have potential to differentiate into fibroblasts, adipocytes, chondrocytes and muscle cells under appropriate circumstances. Osteoprogenitor cells are located on the endosteal and periosteal surface of the bone and inner surface of the Haversian canals (see Fig. 6.7). Some osteoprogenitor cells transform into osteoblasts after being divided by mitosis. Osteoprogenitor cells are activated during the bone remodeling process or regeneration of injury. Some of the transcription factors such as core binding factor alpha-1 and osteoblast-specific genes like bone morphogenic factor (BMP) and transforming growth factor-β (TGF-β) play crucial roles throughout the processes.

Osteoprogenitor cells

Osteoprogenitor cells can arise from stem cells in a variety of tissues. There are as yet no unique identifying markers for the mesenchymal stem cell (MSC) that gives rise to bone, fat, cartilage, and muscle. Bone marrow stroma contains highly proliferative cells that will form single colonies or colony-forming units-fibroblasts (CFU-Fs) and are thought to contain mesenchymal stem cells that can be distinguished from early hematopoietic precursors.62–65 Stromal cells grown in vitro are heterogeneous with respect to the capacity for differentiation, and only a low percentage of all CFU-Fs have stem cell properties.62,63,66,67 Further, only a small fraction of CFU-Fs are bone-forming cells.68,69 Although a specific osteoprogenitor marker has not been identified, antibodies have been developed (e.g. STRO-1, SP-10, SH2, HOP-26) that recognize some subsets of precursor cells.70–77

Mesenchymal differentiation may proceed by a multistep hierarchical process with greater cell lineage restriction to terminal cell types at each subsequent step.78 This idea is supported by identification of bipotential adipocyte-osteoblast precursors, and has suggested that there may be an inverse relationship between adipocyte and osteoblast differentiation.79,80 A related observation is the transdifferentiation of bone to fat cells, or fat to bone cells.81–84 Depending on the local cellular environment, ‘committed’ MSC progeny may also dedifferentiate into another lineage.85 Thus, plasticity may be a common phenomenon; however, at least some reports suggest that cell fusion events may confound this paradigm.86–90 Figure 38.1 shows a simplified scheme for osteoblast differentiation.

MSCs similar to those found in bone marrow have been found in adult peripheral blood, fetal cord blood, fetal liver, tooth pulp, muscle satellite cells, and extramedullary adipose tissue and have osteogenic potential.91–100 Multipotent adult progenitor cells (MAPCs) derived from bone marrow can contribute to most somatic cell types, including skeletal tissue.101,102 Pericytes can also be induced toward the osteogenic lineage.103

7.2 Osteocyte ontogeny

Osteoprogenitor cells residing in the bone marrow give rise to osteoblasts that progress through a series of maturational stages resulting in the mature osteocyte. Biomarkers and functional assays have been used to discriminate between these various stages. Although numerous markers for osteoblasts are available (cbfa1, osterix, alkaline phosphatase, collagen type 1, osteocalcin, etc., see also Chapter 3: Development of the skeleton), only within the past decade have markers have been available for osteocytes. Osteocytes not only share some markers with their progenitors-osteoblasts but also express unique markers based on their morphology and potential function. Markers for osteocytes include phosphate-regulating neutral endopeptidase on the chromosome X (Phex), Dentin Matrix protein 1 (Dmp1), and E11/gp38 for early osteocytes and sclerostin, fibroblast growth factor 23 (Fgf23), and matrix extracellular phosphoglycoprotein (MEPE) for mature osteocytes. These and other markers will be defined later in this chapter.

Kalajzic et al. have used promoters for osteocalcin and collagen type 1 linked to reporters such as green fluorescent protein (GFP) to examine transgene expression during osteoblast differentiation [5]. Osteocalcin-GFP was expressed in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes, whereas GFP driven by the collagen type 1 promoter was strongly expressed in osteoblasts and osteocytes. These investigators also generated an osteocyte-selective promoter, the 8 kb Dmp1 driving GFP that showed expression in osteocytes [6]. This mouse model has proved useful to examine osteocyte ontogeny and to determine osteocyte function. However, there remains a need to identify and generate additional osteocyte promoters not only to drive reporters but also to drive Cre expression.

The differentiating osteoblast has one of three fates; it can become embedded in its own osteoid and continue differentiation into an osteocyte, it can quiesce into a lining cell; or, more likely, it can undergo apoptosis (for review see Manolagas [7]). Karsdal et al. proposed that matrix metalloproteinase activation of latent transforming growth factor β (TGFβ) blocks osteoblast apoptosis thereby delaying differentiation into osteocytes [8]. Identification of mechanisms responsible for osteoblast apoptosis has implications for the development of strategies that could potentially increase bone mass. Inhibition of osteocyte apoptosis may have beneficial or nonbeneficial effects on bone depending on condition as addressed later in this chapter (Fig. 7.2).

BONE CELLS

Osteoprogenitor cells differentiate from mesenchymal stem cells in bone marrow. Bone marrow can, therefore, be seen as a reservoir of osteoprogenitor cells, which differentiate further into pre-osteoblasts, which then develop into mature osteoblasts on reaching the substrate onto which bone will be deposited. There are numerous osteoprogenitor cells in the marrow of young, growing individuals, but either their number or their potential to differentiate further to mature osteoblasts declines considerably with age. The origin of osteoblasts recruited for bone synthesis later in life remains unclear, but there is evidence that these stem cells are also present in the membranes surrounding bones—the periosteum and endosteum (Figure 2-1). Osteoblasts are responsible for the formation of osteoid, the unmineralized component of bone tissue. Osteoblasts contain the normal basic ingredients of a cell-a single nucleus, endoplastic reticulum, well-developed Golgi bodies, and numerous ribosomes and mitochondria—reflecting the requirement for abundant protein synthesis in osteoid formation (Figure 2-2). These proteins include pro-α collagen (a principle component of collagen), osteocalcin, and bone morphogenic proteins. Differentiation of osteoblasts is stimulated by transforming growth factor and bone morphogenic proteins. These components are activated by chemical signals from local cells and serum hormones such as vitamin D metabolites and parathyroid hormone (PTH). The lifespan for osteoblasts is typically around 6 months and 10–15% of them become surrounded by bone and differentiate into osteocytes, while others transform into bone lining cells.

Osteocytes originate as osteoblasts that have become embedded in bone matrix secreted by surrounding osteoblasts. They are smaller than osteoblasts and have fewer of the protein-forming organelles because they are not involved in matrix formation. They do, however, retain the fine threadlike cytoplasmic extensions of osteoblasts that extend to communicate with surrounding osteocytes, bone lining cells, and osteoblasts, forming a network or syncytium. Osteocytes reside in almond-shaped cavities within the bone matrix that are called lacunae (Figure 2-3) and their cytoplasmic extensions pass though small channels in the bone matrix called canaliculi, of which a single osteocyte may typically have 50–70. This network of canaliculi also permits the transport of nutrients, waste products, and chemical signals between blood vessels and bone cells. Osteocytes comprise approximately 90% of all cells in mature bone. During the initial stage of its life cycle, the osteocyte appears to be involved in the process of final or secondary mineralization of adjacent matrix (see subsequent text). The process of secondary mineralization by osteocytes is much slower, in part, because there are many fewer cells per cubic millimeter of tissue than is the case during osteoid formation and primary mineralization by osteoblasts. Another factor may be that mineral ions, cell nutrients, and cell metabolites must pass through the canalicular system. The process of secondary mineralization not only takes longer than primary mineralization (many months versus hours to days), but becomes even slower with increasing age. This results in greater variability in mineral density between mature and more recent bone tissues in older individuals. This will be discussed later in the section on osteon remodeling.

There is growing evidence that the mechanical loading of bone is sensed by osteocytes via extracellular fluid being forced through the network of canaliculi. This fluid, which carries charged ions, generates streaming potentials that stimulate the osteocytes and they in turn send electrochemical or hormonal signals to other cells involved in bone maintenance and remodeling. Osteocytes also appear to regulate the flow of calcium and phosphorus into and out of bone matrix under stimuli from thyroid hormone (calcitonin) and PTH. Osteocytes may live for several years before being replaced as part of the normal mechanism of bone remodeling, which may in some instances be stimulated by osteocyte apoptosis or programmed cell death.

When active bone synthesis ceases at a particular site the osteoblasts enter a quiescent phase, in that they flatten out and become bone lining cells that enclose the bone surface. Bone lining cells have fewer organelles than osteoblasts, reflecting the reduced demand for protein synthesis. They do, however, perform an important function in preventing bone resorption by osteoclasts.

Osteoclasts are responsible for the breakdown and resorption of bone tissue. They differ markedly from osteoblasts in that they are very large, irregularly shaped cells with more than one nucleus (Figure 2-4). There are no cytoplasmic extensions from the cell, which contains many more lysosomes, reflecting its potential for destruction of bone tissue. Mitochondria and the endoplasmic reticulum are minor components, indicating that protein synthesis by osteoclasts is minimal. Osteoclasts arise from the fusion of several blood-borne mononuclear macrophage precursors, which in turn are derived from hematopoetic stem cells in bone marrow under stimulation from vitamin D3 (see subsequent text). They are very active, motile cells and move around the resorbing surfaces of bone. Furthermore, osteoclasts frequently form clusters of cells during resorption and in histological sections of bone several may be seen occupying eroded depressions in the surface known as Howship's lacunae (Figure 2-5). Osteoclasts adhere to bone surfaces via intracellular contractile proteins attached to integrins, which are specialized cell surface receptors (Vaes 1988). This leads the mature osteoclast to form a ruffled border that allows a high surface area in contact with the bone surface. The depressions or lacunae formed by one cell may be removed by the activity of another osteoclast. This may complicate interpretation of bone osteoclast activity, but Howship's lacunae are almost always present if bone resorption has occurred relatively recently before death. Since these cells are absent in archeological bone specimens, one microscopic indicator of active bone resorption is the presence of Howship's lacunae.

Osteoclastic bone resorption involves both mineral dissolution and enzymatic degradation of the organic phase. Hydrogen ion pumps localize to the ruffled border and act with intracellular carbonic anhydrase II to lower the pH of the extracellular bone compartment, forming a resorption pit. This acid environment solubilizes the bone apatite crystals and exposes the organic matrix that is then digested by lysosomal enzymes. The resorption phase is followed by a reversal phase that involves detachment of the osteoclast from the resorbing surface, which is followed by apoptosis or programmed cell death. The exposed surface attracts differentiated osteoblasts that then adhere to the surface and proceed to lay down new osteoid.

Regulation of osteoclastic bone resorption involves a complex web of systemic and local chemical signals. Osteoclasts have specific calcitonin receptors that are induced by PTH, the vitamin D metabolite calcitriol [1,25-(OH)2D3], tumor necrosis factor, and nitric oxide. However, the bone-resorbing hormones PTH and calcitriol are unable to stimulate osteoclasts in the absence of osteoblasts, suggesting that the cellular activity involved in bone resorption and deposition are coupled in some way.

It should be stressed at this point that the cells involved in bone growth, maintenance, and remodeling are influenced by signals from the endocrine, paracrine, and immune systems (Skjodt and Russell 1993). Certain diseases can influence bone remodeling via these systems and their actions on osteoclasts and osteoblasts even though they may have no direct affect on bone tissues. Bone destruction in pathology is, therefore, mediated by osteoclasts (and perhaps monocytes) and all of the destructive lesions seen in skeletal disease are the direct result of osteoclastic action. In an infectious disease such as tuberculosis, the pathogenic organisms stimulate conditions that result in bone destruction by osteoclasts, but do not directly destroy the bone tissue.

2.2 Cell Implantation and Interactions

An osteoprogenitor supply is essential for the niche to function and produce functional bone.35 Because of the chemotactic signals present in the blood and bone tissues, MSCs do themselves migrate to the repair site. In the case of autograft, the graft itself delivers osteogenic and progenitor cells in the form of osteoblasts and bone lining cells capable of mitosis. Therefore, replicating this feature seems a reasonable approach. The addition of such cells may provide the niche an opportunity to accelerate the healing process by circumventing the delay for MSC migration and proliferation. This may also account for autograft's improved healing timeline when compared with approaches without supplementary cells.

Because of the limited availability of multipotent mesenchymal cells, in vitro culture has been anticipated as a useful process for the expansion of cell numbers. Cellular strategies have included the harvest of stem cells from typically bone marrow and either direct implantation or expansion and manipulation in culture environments. The classical approach of tissue engineering, with cell harvest and in vitro expansion, has previously seemed promising36,37 and also considered to be necessary for optimal cell use.38 Possibly because of the additional processes involved, lack of standardization, donor variability, and overall slower progression for bone to date,39–41 such approaches have yet to find mainstream application within bone niche engineering. This also may be partly because of the potential loss of stem cell characteristics in vitro. Bone marrow has been well established as a source of MSC, which has the capability to differentiate down adipogenic, chondrogenic, and osteogenic lineages.1,37 Despite their inherent potential, normal culturing conditions have been associated oxidative damage to the DNA, resulting in MSC senescence, limiting the expansion capability, and reducing the prospective differentiation potential necessary for clinical orthopedic applications.42,43

Other stem cell sources, such as adipose-derived mesenchymal stem cells, have also been reported to have the potential to be redirected in vitro and differentiate down osteogenic lineages. A major benefit over bone marrow sources is the higher number of MSCs being available for harvest,44 making this a potentially effective cell source. However, because of the reported specific molecular phenotype and differences in the differentiation capacity of the adipogenic tissue-derived MSC, this more abundant cell source is often considered suboptimal, despite the greater cell numbers. Importantly, the source location of MSC should be based on their efficacy, and not availability.45 As such, adipocyte-derived MSCs have not gained widespread use to date, though current and future research may change that attitude.

As the periosteum is involved itself in bone regeneration, healing, and growth, the use of this connective tissue provides yet another option within the bone-healing niche. The inner cambium layer of the periosteum is able to differentiate down both osteogenic and chondrogenic lineages,46 with late passage-cultured periosteal cells reported to have a preference for osteogenic marker expression.47 The efficacy of this cell source is suggested to be comparable with bone marrow–derived stem cells, with the location of this cell source being more easily accessible for harvesting. The combination of calcium phosphates and periosteum-derived cells has been shown to support predominately intramembranous bone formation, though this effect is also dependent on the calcium phosphate used.48 In addition, the periosteum may contribute to the paracrine signaling within the niche, such as has been shown with BMP-2 production.49 Apart from osteoblasts, MSC, and pericytes, approximately 90% of the cells in the periosteum are fibroblasts,50 thus making the harvesting and isolation protocols an important consideration for periosteum-related strategies.

Allogenic MSCs have also previously been considered potential candidates to derive suitable osteoprogenitor cells, though their use can elicit unfavorable host responses, necessitating immunosuppressive therapies.51 The use of such cells also has the potential for disease transmission, and therefore has not developed into a revolutionary approach.

An alternative approach is using the beneficial properties of other relevant cell types. Satellite cells, residing in muscle, share a common mesodermal lineage history as osteoprogenitors. These muscle stem cells may provide a further source of both chondrogenic and osteogenic growth factors and stem cells, which can positively interact with periosteal residing osteoprogenitor cells, thus promoting bone healing.52 As bleeding from surrounding soft tissue naturally occurs around many bone defects, this source may hold reasonable relevance compared with other sources. Such surgical approaches, in which muscle flaps are considered, although promising, require closer communication between engineered solutions and surgical approaches to be fully realized.

The use of embryonic stem cells (ESCs), obtained from an embryo at the blastocyst stage, has often been met with controversy and resistance. Theoretically, because of their pluripotency and self-renewal capability, these cells would be potential candidates for bone tissue engineering approaches.53 It has been suggested that ESCs that have undergone in vitro osteogenic differentiation before implantation perform similarly to MSCs for bone formation.54 Conversely, it has also been reported that ESCs are inferior to osteoblasts and MSCs in creating a material resembling the biological and mechanical properties of native bone.55,56 This may be a downside of the pluripotent nature of the ESC, in which in vivo bone tissue formation relies on the ESC differentiation toward osteogenic phenotypes, in which MSCs may more readily follow. ESCs could potentially be more useful for endochondral bone formation,57 characterized by a cartilaginous phase and requiring chondrogenic potential. Induced pluripotent cells, which are derived from adult stem cells, provide another avenue to pluripotent cells. As with many new approaches, the use of induced pluripotent cells requires a regulated approach, and more research is needed to realize their application for bone repair.58 Overall, because of the presence and efficacy of MSCs for bone, it remains to be seen whether the application of pluripotent cells is truly essential to pursue within the bone niche.

Although the use of multipotent stem cells for bone regeneration is still in its infancy, the plethora of alternatives seem to be more easily accessible, and importantly, have the desired standards for safety and acceptability. It may be that the use of cells has greater value in studying in vitro mechanistic and developmental processes, rather than having direct clinical applications. Nonetheless, ongoing research into cell implantation strategies to control the niche remains an encouraging possibility, especially in combination with signals and scaffolds. Their use without in vitro manipulation, such as is the case with bone marrow aspirates, is likely to have the most immediate clinical impact.

Hedgehog signaling in bone and periodontium

Another class of osteoprogenitors have been identified with a Gli1-CreERt2 model by lineage tracing and are also involved in fracture repair (Guo et al., 2018). These Gli1+ stem cells highly expressed CD146, αSMA, and leptin receptor but expressed low levels of Sca1 and NG2. In addition, the conditional removal of the smoothened receptor with Gli1-CreERt2, the key signaling receptor for hedgehog signaling (hh), decreased trabecular bone formation. Also, deletion of β-catenin with this Gli1-CreERt2 model decreased bone formation, indicating that this pool of stem cells is regulated by Wnt signaling. As related to the periodontium, Zhao et al. demonstrated that sonic hedgehog from the neurovascular bundle niche supports mesenchymal stem cell (MSC) differentiation in and around the mouse incisor (Zhao et al., 2014). Recently, these Gli+ cells have also been shown to be the key driver of bone marrow fibrosis in various pathological, hematological, and nonhematological conditions, similar to leptin receptor+ cells (Kramann et al., 2015).

In the periodontium, a critical target associated with periodontitis is the loss of attachment of PDLs to the cementum on the tooth surface and alveolar bone. This enthesis region of the periodontium has many of the same properties as the tendon/ligament attachment to bone and its biology. As discussed above, GDF5 treatment in a primate model has been shown to regenerate not only the bone but also healthy PDLs (Emerton et al., 2011). Gdf5-positive progenitors in the tendon–bone interface give rise to fibrocartilage that mineralizes by hh signaling to form the enthesis (Dyment et al., 2015). Similar mechanisms with hh are suggested to play a key role in enthesis formation in the periodontium. Analysis of the enthesis region in the periodontium has shown selective enrichment of CD31 endothelial cells with attached CD146 stem cells. As the CD146 cells migrate from the vascular niche, they increase the expression of the pericyte NG2 marker near the bone–PDL enthesis region. GDF5 is likely to play a key role in this process as in tendon–bone enthesis biology (Lee et al., 2015).

A key role is played by hh in fracture repair and most likely in periodontium healing. During stress fracture healing, mice were given a selective hh pathway inhibitor, vismodegib. A reduction in both woven bone and later-stage lamellar bone formation and vascularization, with a major decrease in Ibsp and other osteoblast markers during healing, was observed (Kazmers et al., 2015). Follow-up studies demonstrate that activation of the hh pathway by systemic agonist, Hh-Ag1.5, during fracture healing in 18-month old female mice, accelerated the fracture repair process (McKenzie et al., 2018). Little is known about hh in the periodontium, except for its role in incisor stem cell biology. However, Ibsp, a key downstream gene, is critical for cementum-enthesis formation, likely regulated by hh, and worth further investigation (Foster et al., 2013a). Previous in vitro cell experiments suggest that hh signaling and Gli2 transcription factors play a role in regulating Bmp2 gene transcription in response to hh signaling (Zhao and el al., 2006). Whether this mechanism plays a role in vivo is unknown.

Fusion site

Arthrodesis depends on ingress of osteoprogenitor and inflammatory cells from the fusion bed and from the surviving bone cells located in the bone graft. Bone healing is greatly affected by the local blood supply along with the availability of osteoprogenitor cells. The fusion bed vascular supply is a source of supportive nutrients, a vehicle for endocrine signals, and a pathway for the recruitment of osteoprogenitor and inflammatory cells. The preparation of the bony surfaces where the graft material is to be placed also plays an important role in determining the outcome of fusion. Decortication allows vascularization of the fusion bed. In addition, it is important to remove avascular tissue such as scar tissue during surgery since a fusion bed with excessive scar tissue is less likely to achieve successful fusion.

Gain-of-function studies using selective androgen receptor modulators in sexually mature animal models

LoF of AR in osteoprogenitor cells (Ucer et al., 2016) or since conception globally (Callewaert et al., 2009) leads to decreases in trabecular bone mass, connectivity, and cortical area as reported earlier. However, the most compelling data set explores GoF paradigms using nonsteroidal, nonaromatizable SARMs and offers the best approach to fully understand the effects of AR ligands in the mature skeleton.

Earlier reports included suggestions of robust activity in male orchidectomized rat models offered by perspective summaries described earlier (Rosen et al., 2002) but these did not provide an opportunity for detailed analysis of the preclinical datasets. However, the most compelling preclinical evidence was offered by Kearbey et al., who showed a dose-dependent gain in trabecular bone mass in female ovariectomized mature rats that were treated with a distinct aryl propionamide SARM (S-4) (Gao et al., 2005a). They showed protection from ovariectomy-induced trabecular bone loss using the S-4 SARM as measured by lumbar spine BMD. This benefit offered by the SARM was dependent on AR signaling, as observed by the reversal of this protection of spine BMD in the arm that combined the S-4 SARM along with the AR antagonist bicalutamide. In addition, they observed enhancements in cortical bone parameters, including cortical width, cortical density, and periosteal circumference at peak doses of 1.0–3.0 mg/kg-day. Collectively, this report provided compelling evidence of the osteoprotective effects of a nonsteroidal SARM that functions as an AR agonist in bone when used immediately after ovariectomy in female SD rats (Kearbey et al., 2007). To further evaluate the potential for an anabolic response, the same group reported deployment of the S-4 SARM in a delayed rat male orchidectomy model. In this model, the orchidectomy induced an expected loss in total whole-body bone mineral content and BMD over the 12-week wasting period. This change in whole-body BMC and BMD was completely recovered after 8 weeks of treatment with S-4 SARM given daily at 10 mg/kg-day. However, there were some modest reductions in osteocalcin levels, which may point to a decrease in bone turnover in these animals (Gao et al., 2005b). It was not clear whether the authors measured favorable changes in markers such as P1NP, which reflect de novo bone formation as reflected by changes in the N-terminus-modified epitopes presented in serum with the synthesis of new collagen molecules placed onto bone (Eastell et al., 2006). Therefore, while these results are encouraging, it is not definitive whether the gains in total bone mineral content and BMD are due to SARM-mediated gains in bone formation. The most compelling report that showcases the direct anabolic action of SARMs was reported by Hanada et al., who utilized a –tetrahydroquinoline derivative to generate an SARM, termed as S-40503 (Hanada et al., 2003). This molecule was shown to be very selective for AR and did not bind ESR1, ESR2, or other nuclear hormone receptors. In this report, they utilized a bilateral orchidectomized male rat that was treated with the S-40503 SARM for 4 weeks 1-day postgonadectomy. They showed increases in femoral BMD that were dose-dependent after the 4-week treatment period. To address direct anabolic action by SARM S-40503, they utilized an ovariectomized female rat model, wherein animals were allowed to lose bone mass for 4 weeks followed by 4 weeks of daily subcutaneous injections with SARM S-40503 (30 mg/kg-day), estrogen (20 μg/kg-day), or DHT (10 mg/kg-day). They measured distal femur cancellous BMD, which was most improved with estrogen followed by SARM S-40503 and DHT to a similar but qualitatively lesser extent. In contrast, they observed a robust increase in cortical BMD that was greater than intact nonovariectomized age-matched animals for both SARM S-40503 and DHT. This gain in cortical bone BMD was not apparent for the estrogen-only arm. Furthermore, they used calcein double-labeling to show direct evidence of an increased mineral apposition rate (MAR μm/day) in response to nonaromatizable DHT and SARM S-40503, which was in contrast to minimal apposition observed with estrogen (no change). Notably, the MAR observed for S-40503-treated animals was higher than for the intact nonovariectomized control, again suggesting direct anabolic action in response to this GoF paradigm using both a nonaromatizable androgen (i.e., DHT) and a nonsteroidal selective SARM, S-40503. Collectively, the use of structurally distinct nonsteroidal SARMs in preclinical models suggests a potential to directly and favorable impact bone mass in animals undergoing gonadectomy-induced bone loss (see Fig. 41.3). However, these preclinical findings have yet to translate to changes in improvements in bone mass or bone biomarkers in clinical studies exploring SARMs. This may be due to the positioning of these SARMs in diseases such as cancer cachexia and sarcopenia, which may require long-term use to determine such bone-related end points.

Soft-Tissue Bed

Spine fusion depends on the influx of osteoprogenitor and inflammatory cells. The local soft tissues must support bone graft healing. An adequate blood supply is a critical requirement for success. Fusion bed vasculature supplies nutrients to the maturing fusion, provides endocrine stimuli, and is a source of inflammatory and osteoprogenitor cells. Nonviable and traumatized tissues should be removed from the graft site.

Hurley et al.70 evaluated the role of local soft tissues in a canine dorsal spine fusion model. Thirty-seven animals underwent a modified Hibbs fusion (control), a Hibbs fusion with a fluid-permeable, cell impermeable membrane interposed between fusion site and muscle mass, or with a membrane impermeable to both cells and fluids. All 12 animals with the semipermeable membrane fused; none of the 10 animals with the impermeable membrane did so.

Radiation has a detrimental effect on a healing spine fusion, especially in the first few postoperative weeks. This effect may be caused by cytotoxicity, but is probably also the product of the resultant intense vasculitis and inhibition of angiogenesis. Even after the acute injury, radiation-induced osteonecrosis and dense, hypovascular scars make for a poor fusion environment. Studies suggest that a 3- to 6-week delay in radiation would be beneficial to the fusion process.15,38 Use of vascularized grafts anastomosed to nonirradiated vessels may also increase the chance of successful fusion.

Soft Tissue Bed

Spine fusion depends on the influx of osteoprogenitor cells, inflammatory cells, nutrients, and endocrine stimuli from the local soft tissues to support bone graft healing. An adequate blood supply in this environment is a critical requirement for success, and nonviable, traumatized tissues should be removed from the graft site.

Hurley and colleagues evaluated the role of these factors in a canine dorsal spine fusion model.85 Thirty-seven animals underwent a modified Hibbs fusion (control), a Hibbs fusion with a fluid-permeable/cell-impermeable membrane interposed between fusion site and muscle mass, or with a membrane impermeable to both cells and fluids. Fusion was achieved in all 12 animals with the semipermeable membrane, whereas no animals with the impermeable membrane had a successful fusion.

Radiation has a detrimental effect on a healing spine fusion that is especially pronounced in the first few postoperative weeks. This effect may be caused by cytotoxicity, but is probably a product of the resultant vasculitis and inhibition of angiogenesis that follows radiation treatment. In the long term, radiation also can induce osteonecrosis and dense hypovascular scars, creating a poor fusion environment. Studies suggest that a 3- to 6-week delay in radiation would be beneficial to the fusion process.91,92 Use of vascularized grafts anastomosed to nonirradiated vessels also may increase the chance of successful fusion.