Monthly Archives: August 2015

How bone knows how to grow

How does bone know how to be the proper shape and size for development?  Can we manipulate this to grow taller?

I received this email from the author regarding how distraction osteogenesis would affect how bone manipulates growth in regards to maintaining placement of superstructures: \

“It is indeed an interesting question as it challenges the system with an unnatural manipulation – i.e. interstitial growth.

The simple answer is: we haven’t tried, so I can’t say for sure.

If the relative locations of ligament and tendon insertions are what you are interested in, then previous works show that the periosteum is involved in regulation of their positions (see list below). Moreover, if the balance between proximal and distal growth rates is what you are interested in, then other works show that cross-sectional cutting and stripping of the periosteum can cause temporal acceleration in overall growth rate of the bone (also in humans, if I remember correctly), followed by a potential change in proximal to distal growth balance (I don’t think that these works test how these influence the positioning of superstructures in the bone; see list below).
Therefore, if the operation you are applying includes anchoring of the periosteum to the bone or its cutting and stripping, this is something that may influence the scaling of the bones.”

Isometric Scaling in Developing Long Bones Is Achieved by an Optimal Epiphyseal Growth Balance.

“One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.”

If we can trick the bone into thinking it’s drifting maybe we can convince it to grow to maintain the position of the superstructure.  For example, dislocating the bone or similar means.

Although the molecular mechanisms regulating each growth plate for different bones are similar the bones still have different elongation rates.

“superstructures, known as bone ridges, tuberosities, condyles, etc., are necessary for the attachment of tendons and ligament as well as for articulation. To perform these functions they are located at specific positions along the bone. Bone superstructures emerge during early skeletogenesis . During growth, bones elongate extensively by advancement of the two growth plates away from the superstructures. It is therefore expected that during elongation, superstructures would remain at their original position near the center of the bone. Nevertheless, the end result is proper spreading of superstructures along the mature bone, which clearly implies the existence of a morphogenetic mechanism that corrects their locations.”

It’d be interesting to see what happens to bone superstructures during distraction osteogenesis.

“An ossified bone is a rigid object and so are the superstructures protruding from it, implying that they cannot be relocated by means of cell migration or proliferation. Therefore, any scaling mechanism must be adapted to overcome these physical restrictions.”<-So we have to make the bone less rigid.

“forelimb bones tend to grow away from the elbow joint, whereas bones in hind limbs tend to grow toward the knee joint.”

” Because the periosteal sheath is stretched over the entire external surface of the bone, including both the superstructures and the growth plates, it can pass to the growth plates signals concerning the relative position of superstructures.”<-Then perhaps we can manipulate longitudinal bone growth by manipulating the periosteal sheath.

“periosteal tension down-regulates growth plate activity, as the higher the tension level, the more inhibited growth plate activity is.  Damaged periosteum forms a scar tissue at the site of destruction. This scar tissue, which anchors the periosteum into the bone, creates an independent tension level near each growth plate. As a result, a new growth balance is formed, which equals the ratio between the distances from the site of the scar to the two ends of the bone, therefore maintaining the relative position of the scar site.. Superstructures can be considered as natural anchoring points for the periosteum into the ossified bone, either due to the insertion of tendons through them into the bone cortex, or by means of steric interference, such as in the tibiofibular junction. This results in a regulatory loop whereby the superstructures determine the tension levels of the two periosteal segments, which control the ratio of growth rates by inhibiting growth plate activity, which in turn maintains the relative position of the superstructure.”

Heterotopic Ossification

Heterotopic ossification is endochondral ossification that occurs outside the bone.  Understanding why it occurs can help us find ways to induce endochondral ossification within the bone.  The biggest issue with inducing a new growth plate in bone is the permissive local environment criteria.  The bone likely has to be degraded in some way to induce a neo-growth plate as the existing bone environment likely puts a constraining factor on growth.

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification.

“Heterotopic ossification (HO) is a debilitating condition defined by the de novo development of bone within non-osseous soft tissues, and can be either hereditary or acquired. The hereditary condition, fibrodysplasia ossificans progressiva is rare but life threatening. Acquired HO is more common and results from a severe trauma that produces an environment conducive for the formation of ectopic endochondral bone. Despite continued efforts to identify the cellular and molecular events that lead to HO, the mechanisms of pathogenesis remain elusive. It has been proposed that the formation of ectopic bone requires an osteochondrogenic cell type, the presence of inductive agent(s) and a permissive local environment. To date several lineage-tracing studies have identified potential contributory populations. However, difficulties identifying cells in vivo based on the limitations of phenotypic markers, along with the absence of established in vitro HO models have made the results difficult to interpret. The purpose of this review is to critically evaluate current literature within the field in an attempt identify the cellular mechanisms required for ectopic bone formation. The major aim is to collate all current data on cell populations that have been shown to possess an osteochondrogenic potential and identify environmental conditions that may contribute to a permissive local environment. This review outlines the pathology of endochondral ossification, which is important for the development of potential HO therapies and to further our understanding of the mechanisms governing bone formation.”

“of the 80 % of war victims who suffer major extremity trauma during combat injury, approximately 64 % of these patients go on to develop some degree of HO”

“Current evidence suggests that the formation of ectopic bone in vivo requires three primary conditions: (1) a cell type capable of osteogenic differentiation, (2) the presence of inductive agents and (3) a permissive local environment”

“o date many contributory biological factors have been implicated in the aetiology, including the bone morphogenetic proteins (BMPs), inflammation, prostaglandin E2, hypercalcemia, hypoxia, abnormal nerve activity, immobilisation and dysregulation of hormones”
heterotropic endochondral ossification factors

“Tissue damage leads to the infiltration of immunological cells (monocytes, neutrophils and leukocytes) through the local vasculature. Resulting fibro-proliferation of an as yet unknown cell population is accompanied by hypoxia and the generation of brown adipose tissue at the site of damage. The presence of adipose tissue is hypothesised to lower the local oxygen tension leading to the establishment of a chondrogenic environment. Neovascularisation accompanies chondrogenesis and provides an avenue through which systemic cell types (endothelial cells, pericytes etc.,) may enter the injury site, and potentially contributed to osteochondrogenic differentiation. A subsequent increase in local oxygen tension promotes chondrocyte maturation and hypertrophy. The collagenous matrix deposited by these cells is then remodelled and ossified to form endochondral bone”<-If we induce such factors in the bone we can create new growth plates in there too.

“MSCs have frequently been shown to form endochondral bone when cultured under appropriate conditions (e.g. under hypoxia and/or in the presence of TGF-β)”

“MSCs may also contribute to chondrocyte hypertrophy and the progression of HO via their immunomodulatory effects, primarily through the production of anti-inflammatory cytokines and nitric oxide (NO)”

Several cell types are listed that are capable of heterotopic ossification are likely present in bone.

“Bone marrow HSC side population    Lin−/Sca-1+/cKit+/CD45+”

“Mesenchymal precursor cell (MPC)    CD44+/CD49e+/CD73+/CD90+/CD105+”

“MSC    CD73+/CD90+/CD105+”

“cells presenting the glutamate transporter GLAST were found to contribute to the formation of ectopic bone, and that these GLAST+ cells appeared to be distinct from the Tie2+ population”

” a significant upregulation in transcriptional activity in key osteogenesis-related genes (ALPL, BMP-2, BMP-3, COL2A1, COLL10A1, COL11A1, COMP, CSF2, CSF3, MMP8, MMP9, SMAD1 and VEGFA) in patients that developed HO compared to those who did not.”

Influence of transcutaneous electrical stimulation on heterotopic ossification: an experimental study in Wistar rats.

“Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues, resulting in joint mobility deficit and pain. Different treatment modalities have been tried to prevent HO development, but there is no consensus on a therapeutic approach. Since electrical stimulation is a widely used resource in physiotherapy practice to stimulate joint mobility, with analgesic and anti-inflammatory effects, its usefulness for HO treatment was investigated. We aimed to identify the influence of electrical stimulation on induced HO in Wistar rats. Thirty-six male rats (350-390 g) were used, and all animals were anesthetized for blood sampling before HO induction, to quantify the serum alkaline phosphatase. HO induction was performed by bone marrow implantation in both quadriceps of the animals, which were then divided into 3 groups: control (CG), transcutaneous electrical nerve stimulation (TENS) group (TG), and functional electrical stimulation (FES) group (FG) with 12 rats each. All animals were anesthetized and electrically stimulated twice per week, for 35 days from induction day. After this period, another blood sample was collected and quadriceps muscles were bilaterally removed for histological and calcium analysis and the rats were killed. Calcium levels in muscles showed significantly lower results when comparing TG and FG (P<0.001) and between TG and CG (P<0.001). Qualitative histological analyses confirmed 100% HO in FG and CG, while in TG the HO was detected in 54.5% of the animals. The effects of the muscle contractions caused by FES increased HO, while anti-inflammatory effects of TENS reduced HO.”

“The formation of heterotopic bone may be due to muscle trauma (myositis ossificans). It is common in people who have undergone total hip arthroplasty , those with spinal cord injuries, and victims of head trauma, all of which often lead to long periods of immobilization of the affected limbs.”

“skeletal muscle serves as a physical safeguard for the other organs and is anatomically located immediately beneath the skin, so it represents the most damaged organ in the body. Although skeletal muscle is characterized by the presence of fatty and connective tissues that originated from nonmyogenic mesenchymal progenitors, those progenitors were initially identified in BM”

“Muscle contraction occurs by the deposition of calcium in muscle tissue, and this stimulates the sliding of actin and myosin myofibrils, which characterizes the contractile process”

“electrical stimulation helps the deposition of calcium, causes changes in oxygen content and pH, stimulates expression of growth factors, and recruits help in osteoblast migration and secretion of extracellular matrix (ECM), leading to bone formation.”

“Mechanotransduction refers to the process by which the body converts a mechanical stimulus into a cellular response”

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis.

“Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

LDL receptor deficient (LDLr−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr−/− mice). Synovial wash-outs of LDLr−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.”

“multiple injections of members of the TGF-super family, such as TGF-β or BMP-2, directly into the knee joint of the mouse caused abundant enthesophyte/osteophyte formation”