Information

Difference in callus formation during healing of bones of different origin


My study material from pathology states that during fracture healing after hemorrhage and coagulum formation the mesenchymal progenital cells from periostium that are closer to the fracture differentiate into chondrocytes and form hyaline cartilage, while the ones that are farther differentiate into osteoblast.

Result is production of hyaline cartilage in which enchondrial ossification and subsequent remodeling into lamelar bone occurs.

My question is: Does this enchondrial ossification also occur in bones in which Intramembranous ossification normally occur during development? (e.g. clavicle and some skull bones.)

NOTE: in this stackexchange post and Wikipedia article on this topic, only chondrocytes are noted.


Bone healing

Bone healing, or fracture healing, is a proliferative physiological process in which the body facilitates the repair of a bone fracture.

Generally bone fracture treatment consists of a doctor reducing (pushing) displaced bones back into place via relocation with or without anaesthetic, stabilizing their position to aid union, and then waiting for the bone's natural healing process to occur.

Adequate nutrient intake has been found to significantly affect the integrity of the fracture repair. [1] Age, bone type, drug therapy and pre existing bone pathology are factors which affect healing. The role of bone healing is to produce new bone without a scar as seen in other tissues which would be a structural weakness or deformity. [2]

The process of the entire regeneration of the bone can depend on the angle of dislocation or fracture. While the bone formation usually spans the entire duration of the healing process, in some instances, bone marrow within the fracture has healed two or fewer weeks before the final remodeling phase. [ citation needed ]

While immobilization and surgery may facilitate healing, a fracture ultimately heals through physiological processes. The healing process is mainly determined by the periosteum (the connective tissue membrane covering the bone). The periosteum is one source of precursor cells which develop into chondroblasts and osteoblasts that are essential to the healing of bone. Other sources of precursor cells are the bone marrow (when present), endosteum, small blood vessels, and fibroblasts. [3]


Bone Remodeling and Repair

Bone renewal continues after birth into adulthood. Bone remodeling is the replacement of old bone tissue by new bone tissue. It involves the processes of bone deposition or bone production done by osteoblasts and bone resorption done by osteoclasts, which break down old bone. Normal bone growth requires vitamins D, C, and A, plus minerals such as calcium, phosphorous, and magnesium. Hormones such as parathyroid hormone, growth hormone, and calcitonin are also required for proper bone growth and maintenance.

Bone turnover rates, the rates at which old bone is replaced by new bone, are quite high, with five to seven percent of bone mass being recycled every week. Differences in turnover rates exist in different areas of the skeleton and in different areas of a bone. For example, the bone in the head of the femur may be fully replaced every six months, whereas the bone along the shaft is altered much more slowly.

Bone remodeling allows bones to adapt to stresses by becoming thicker and stronger when subjected to stress. Bones that are not subject to normal everyday stress (for example, when a limb is in a cast) will begin to lose mass.

Figure (PageIndex<1>): Stages of fracture repair: The healing of a bone fracture follows a series of progressive steps: (a) A fracture hematoma forms. (b) Internal and external calli form. (c) Cartilage of the calli is replaced by trabecular bone. (d) Remodeling occurs.


Late complications of fractures

Local

  • Delayed union (fracture takes longer than normal to heal).
  • Malunion (fracture does not heal in normal alignment).
  • Non-union (fracture does not heal).
  • Joint stiffness.
  • Contractures.
  • Myositis ossificans [5] .
  • Avascular necrosis.
  • Algodystrophy (or Sudeck's atrophy). .
  • Growth disturbance or deformity.

Systemic

Problems with bone healing (non-union, delayed union and malunion)

Delayed union is failure of a fracture to consolidate within the expected time - which varies with site and nature of the fracture and with patient factors such as age. Healing processes are still continuing, but the outcome is uncertain.

Non-union occurs when there are no signs of healing after >3-6 months (depending upon the site of fracture). Non-union is one endpoint of delayed union. The distinction between delayed union and non-union can be slightly arbitrary: whilst fractures can generally be expected to heal in 3-4 months, this will vary in the case of open fractures and those associated with vascular injury, and also in the presence of patient risk factors described below. However, non-union is generally said to occur when all healing processes have ceased and union has not occurred.

Malunion occurs when the bone fragments join in an unsatisfactory position, usually due to insufficient reduction.

Factors predisposing to delayed union [6]

  • Severe soft tissue damage.
  • Inadequate blood supply.
  • Infection.
  • Insufficient splintage.
  • Excessive traction.
  • Older age.
  • Severe anaemia. . level. .
  • Medications including NSAIDs and steroids.
  • Complicated/compound fracture. .

Factors disposing to non-union
Delayed union and non-union occurs in approximately 5-10% of all fractures but is more common in open long bone fractures (17% non-union) or where there is motion at the fracture site [7] . Risk factors are all of those above and also:

  • Too large a space for bony remodelling to bridge.
  • Interposition of periosteum, muscle or cartilage.
  • Bony site with a limited blood supply: some sites are more vulnerable to compromise of blood supply by the fracture (eg, scaphoid, femoral head and neck, and tibia).

Presentation of non-union

  • Pain at fracture site, persisting for months or years.
  • Non-use of extremity.
  • Tenderness and swelling.
  • Joint stiffness (prolonged >3 months).
  • Movement around the fracture site (pseudarthrosis).
  • Palpable gap at fracture site.
  • Absence of callus (remodelled bone) or lack of progressive change in the callus suggests delayed union.
  • Closed medullary cavities suggest non-union.
  • Radiologically, bone can look inactive, suggesting the area is avascular (known as atrophic non-union) or there can be excessive bone formation on either side of the gap (known as hypertrophic non-union).

Management of non-union
Non-surgical approaches:

  • Early weight bearing and casting may be helpful for delayed union and non-union.
  • Bone stimulation can sometimes be used. This delivers pulsed ultrasonic or electromagnetic waves to stimulate new bone formation. It needs to be used for up to an hour every day, and may take several weeks to be effective.
  • Medical treatments such as teriparatide have also been used to promote fracture healing, particularly in patients with osteoporosis [7] .
  • Debridement to establish a healthy infection-free vascularity at the fracture site.
  • Bone grafting to stimulate new callus formation. Bone may be taken from the patient or may be cadaveric.
  • Bone graft substitutes/osteobiologics.
  • Internal fixation to reduce and stabilise the fracture. (Bone grafting provides no stability.)
  • Depending on the type of non-union, any combination of the above [6] .

Myositis ossificans

Myositis ossificans occurs when calcifications and bony masses develop within muscle and can occur as a complication of fractures, especially in supracondylar fractures of the humerus [5] . The condition tends to present with pain, tenderness, focal swelling, and joint/muscle contractures. Avoid excessive physiotherapy rest the joint until pain subsides NSAIDs may be helpful and consider excision after the lesion has stabilised (usually 6-24 months). It may be difficult to distinguish from osteogenic sarcoma [5] .

Algodystrophy

Algodystrophy, also known as Sudeck's atrophy, is a form of reflex sympathetic dystrophy (or complex regional pain syndrome type 1), usually found in the hand or foot. More than 40% of reflex sympathetic dystrophies follow trauma, notably fractures [8] . A continuous, burning pain develops, accompanied at first by local swelling, warmth and redness, progressing to pallor and atrophy. Movement of the afflicted limb is very restricted. Treatment is usually multi-pronged:

  • Rehabilitation - physiotherapy and occupational therapy to decrease sensitivity and gradually increase exercise tolerance.
  • Psychological therapy.
  • Pain management - often difficult and with a disputed evidence base. Approaches used are neuropathic pain medications (eg, amitriptyline, gabapentin, opioids), steroids, calcitonin, intravenous bisphosphonates and regional blocks.

Iatrogenic complications of fracture treatment

Casts
Poor cast placement may lead to problems of malunion, either because the bones are not accurately aligned or because the fracture is not sufficiently immobilised.

Prolonged cast immobilisation, or 'cast disease', can create circulatory disturbances, inflammation, and bone disease resulting in osteoporosis, chronic oedema, soft tissue atrophy, and joint stiffness. Good physiotherapy will help avoid these problems. Casts may also cause:

Patients need clear information on managing a cast - for example, on keeping it dry, on reporting increased pain or tingling/numbness. Sharp edges rubbing on the skin may need to be trimmed or filed. Poor cast management leading to wetness of the skin beneath the cast can affect skin integrity, which increases the risk of infection.

Casts lead to some loss of bone density in the affected limb, a phenomenon which is seen regardless of the type of casting or skill involved [9] .

Traction
Traction prevents patients mobilising, causing additional muscle wasting and weakness. Other complications of traction include:

  • Pressure ulcers.
  • Pneumonia/urinary tract infections.
  • Permanent footdrop contractures.
  • Peroneal nerve palsy.
  • Pin tract infection.
  • Thromboembolism.

External fixation
Problems caused by external fixation include:

  • Pin tract infection.
  • Pin loosening or breakage.
  • Interference with movement of the joint.
  • Neurovascular damage due to pin placement.
  • Misalignment due to poor placement of the fixator.
  • Psychological complications: external fixation can have a massive psychological impact on the patient. Altered body image and a sense of visible disability, deformity or mutilation can occur. Some patients have to adjust their device and assist with pin site care, and this may also be frightening. Provision of adequate information before fixation, where possible, and support and information after the procedure are an essential part of care.

Further reading and references

Non Union Wheeless' Textbook of Orthopaedics

Ingoe HM, Coleman E, Eardley W, et al Systematic review of systematic reviews for effectiveness of internal fixation for flail chest and rib fractures in adults. BMJ Open. 2019 Apr 19(4):e023444. doi: 10.1136/bmjopen-2018-023444.

de Bruijn JA, van Zantvoort APM, van Klaveren D, et al Factors Predicting Lower Leg Chronic Exertional Compartment Syndrome in a Large Population. Int J Sports Med. 2018 Jan39(1):58-66. doi: 10.1055/s-0043-119225. Epub 2017 Nov 10.

Gonzalez Quevedo D, Sanchez Siles JM, Rojas Tomba F, et al Blisters in Ankle Fractures: A Retrospective Cohort Study. J Foot Ankle Surg. 2017 Jul - Aug56(4):740-743. doi: 10.1053/j.jfas.2017.02.003.

Uransilp N, Muengtaweepongsa S, Chanalithichai N, et al Fat Embolism Syndrome: A Case Report and Review Literature. Case Rep Med. 2018 Apr 292018:1479850. doi: 10.1155/2018/1479850. eCollection 2018.

Meyers C, Lisiecki J, Miller S, et al Heterotopic Ossification: A Comprehensive Review. JBMR Plus. 2019 Feb 273(4):e10172. doi: 10.1002/jbm4.10172. eCollection 2019 Apr.

Andrzejowski P, Giannoudis PV The 'diamond concept' for long bone non-union management. J Orthop Traumatol. 2019 Apr 1120(1):21. doi: 10.1186/s10195-019-0528-0.

Kostenuik P, Mirza FM Fracture healing physiology and the quest for therapies for delayed healing and nonunion. J Orthop Res. 2017 Feb35(2):213-223. doi: 10.1002/jor.23460. Epub 2016 Dec 19.

Guthmiller KB, Varacallo M Complex Regional Pain Syndrome (CRPS), Reflex Sympathetic Dystrophy (RSD)


Foot Injuries

Stephen M. Simons , Robert Kennedy , in Clinical Sports Medicine , 2007

Calluses

Calluses are thickening of the skin in areas of pressure, friction or other irritation on the plantar foot. They may indicate prominent bones such as calluses over low riding metatarsal heads. A crescent shaped callus on the medial side of the second metatarsal head indicates inadequate weight acceptance and often a hypermobile first ray. The second metatarsal bears undue pressure. The callous is crescent shaped, apex directed toward the first ray, and usually distal to the MTP indicating terminal stance phase shear forces to the skin. Inappropriate footwear will also lead to callus formation.

Typical therapy utilizes padding around smaller calluses to relieve the pressure and pain. Doughnut shaped or ‘U’-shaped padding can be cut from thin foam padding. Orthotics with cutouts for low riding metatarsal heads or Morton's extension for hypermobile first ray corrects mechanical shortcomings. Salicylic acid pads help to shrink a callus and debridement provides an immediate reduction in the callus size and pain.

Despite these treatments, the intractable plantar keratoses will persist. Prominent plantar condyles on the metatarsal head or abnormally plantar directed metatarsals predispose to this problem. Surgery may be required to elevate the distal metatarsal or remove the plantar condyles of the metatarsal heads.


Show/hide words to know

Chondroblasts: cell that make cartilage and help in bone healing after a break.

Hard callus: a hard bump that forms around a fracture when a bone is broken and healing.

Osteoclast: cells in your body that break down bone material in order to reshape it.

Phagocytes: cells that swallow up germs and other unwanted waste materials in the body.

Soft callus: a soft bump that forms around a fracture when a bone is broken and healing.


Scaffolds and bone substitutes

Although they lack osteoinductive or osteogenic properties, synthetic bone substitutes and biomaterials are already widely used in clinical practice for osteoconduction. DBM and collagen are biomaterials, used mainly as bone-graft extenders, as they provide minimal structural support [8]. A large number of synthetic bone substitutes are currently available, such as HA, β-TCP and calcium-phosphate cements, and glass ceramics [8, 23]. These are being used as adjuncts or alternatives to autologous bone grafts, as they promote the migration, proliferation and differentiation of bone cells for bone regeneration. Especially for regeneration of large bone defects, where the requirements for grafting material are substantial, these synthetics can be used in combination with autologous bone graft, growth factors or cells [8]. Furthermore, there are also non-biological osteoconductive substrates, such as fabricated biocompatible metals (for example, porous tantalum) that offer the potential for absolute control of the final structure without any immunogenicity [8].

Research is ongoing to improve the mechanical properties and biocompatibility of scaffolds, to promote osteoblast adhesion, growth and differentiation, and t0 allow vascular ingrowth and bone-tissue formation. Improved biodegradable and bioactive three-dimensional porous scaffolds [55] are being investigated, as well as novel approaches using nanotechnology, such as magnetic biohybrid porous scaffolds acting as a crosslinking agent for collagen for bone regeneration guided by an external magnetic field [56], or injectable scaffolds for easier application [57].


Abstract

Fractures are the most common large-organ, traumatic injuries to humans. The repair of bone fractures is a postnatal regenerative process that recapitulates many of the ontological events of embryonic skeletal development. Although fracture repair usually restores the damaged skeletal organ to its pre-injury cellular composition, structure and biomechanical function, about 10% of fractures will not heal normally. This article reviews the developmental progression of fracture healing at the tissue, cellular and molecular levels. Innate and adaptive immune processes are discussed as a component of the injury response, as are environmental factors, such as the extent of injury to the bone and surrounding tissue, fixation and the contribution of vascular tissues. We also present strategies for fracture treatment that have been tested in animal models and in clinical trials or case series. The biophysical and biological basis of the molecular actions of various therapeutic approaches, including recombinant human bone morphogenetic proteins and parathyroid hormone therapy, are also discussed.


Skeletal Stem and Progenitor Cells in Developing Bone

Different genetic markers and/or various other approaches have been utilized to characterize several populations of skeletal stem cells in fetal, neonatal or early postnatal bones. Some of these have already been mentioned above (e.g., Prx1+, Gli1+, Grem1+, Nestin+) and others include Sox9+ SSPCs (Akiyama et al., 2005 He et al., 2017), Col2+ SSPCs (Ono et al., 2014b), Osx+ SSPCs (Greenbaum et al., 2013 Mizoguchi et al., 2014 Tzeng et al., 2018) and Lin-AlphaV+CD200+ SSPCs (Short et al., 2009). Genetic labeling reveals that some of these give rise to others during development (i.e., cells expressing Prx1 begin expressing Osx, Sox9, and Col2), while other populations overlap partially (i.e., Sox9+ and Col2+ cells, Osx+ and Prx1+ cells). In addition, essentially every mouse strain mentioned above targets perichondrium (i.e., Prx1, Nestin-GFP, Sox9, Col2, Grem1, Gli1, and Osx markers) and/or the growth plate chondrocytes (i.e., Prx1, Sox9, Col2, Gremlin, and Gli1 markers). It is important to emphasize that during development the perichondrium gives rise to bone marrow stroma (Maes et al., 2010) and, as discussed above, retains SSPCs into adulthood (Yang et al., 2013 Debnath et al., 2018). Moreover, trans-differentiation of hypertrophic chondrocytes of the growth plate into various mesenchymal-type cells of the bone marrow is well established (Yang G. et al., 2014, Yang L. et al., 2014 Zhou X. et al., 2014) and particularly intensive during the early stages of longitudinal bone growth (Li et al., 2017 Mizuhashi et al., 2018 Newton et al., 2019).

The potential influx of new stem/progeny cells from other sources [e.g., Schwann and endoneurial fibroblasts (Carr et al., 2019 Xie et al., 2019b)] makes the development of the skeleton even more complex. From our perspective, the information presently available is indicative of phenotypic plasticity of cells of mesenchymal original and high developmental dynamics during neonatal growth. FACS-based characterization of SSPCs such as Lin-AlphaV+CD200+ SSPCs (Chan et al., 2015, 2018) obtained from surgical samples of fetal or neonatal growth plate surrounded by innervated perichondrium may resolve this issue by identifying bona fide skeletal stem cells. However, this approach does not reveal their exact location or the nature of their microenvironment and allows only limited manipulation of these cells in their natural milieu.

Of course, every population of SSPCs identified provides valuable information, but, at the same time, little insight can be made into location or composition of the niche during this period. Indeed, in vivo identification and localization of the progeny of any specific type of SSPCs is virtually impossible in this dynamic setting. Nevertheless, one pattern is becoming clear.

Several markers – including Grem1 (Worthley et al., 2015), Gli1 (Shi et al., 2017), LepR (Zhou B.O. et al., 2014), Osx (at its multipotency stage, Mizoguchi et al., 2014), PDGFRβ (Böhm et al., 2019), Prx1 (Greenbaum et al., 2013), Nestin-GFP (Ono et al., 2014a), and Col2 (Ono et al., 2014b) – reveal the presence of putative stem cells in the region of the primary spongiosa, immediately below the growth plate. The primary spongiosa is a unique area characterized by intensive bone formation and tissue remodeling. Its distinct extracellular matrix is comprised of remnants of calcified cartilage enriched in type X collagen and osteopontin and containing high levels of matrix metalloproteinases such as MMP9, MMP13, and MMP14, highly active osteoclasts, active trans-differentiation of hypertrophic chondrocytes, active angiogenesis and unique arrangements of endothelial cells into hemospheres (Wang et al., 2013).

Furthermore, hypertrophic chondrocytes express a variety of cytokines, including vascular endothelial growth factor (VEGF), RANKL, OPG, and Ihh (Houben et al., 2016), which also may participate in creating a proper microenvironment for SSPCs. For example, ablation of Ihh in the growth plate employing Col2-Cre attenuates both Wnt signaling and the maturation of osteoblasts within the primary spongiosa (Maeda et al., 2007). Thus, this combination of features may be the key to creating and maintaining the SSPC niche in growing bones.

However, detailed determination of the components of this niche and their roles in the regulation of individual populations of SSPCs within the primary spongiosa will probably require approaches that are more advanced and sophisticated than those been utilized to date. Moreover, it is important to remember that in humans the growth plate fuses (disappears) during late puberty, in association with the cessation of growth, whereas in mice the growth plate remains open while these animals continue growing into adulthood (Emons et al., 2011 Chagin and Newton, 2019). Accordingly, findings on mice must be applied to humans only with care.


Discussion

In this study, the effects of cyclic purely axial compression amplitudes on bone formation after callus distraction were investigated. The results showed that the larger compression amplitude (Group 0.6 mm) representing moderate compressive interfragmentary movements (IFM) stimulated significantly more new bone formation than the small compression amplitude (Group 0.1 mm), but in both groups only intramembranous bone formation was observed.

The finding that moderate predominantly compressive amplitudes stimulate more bone formation than small compression amplitudes or rigid fixation is in agreement with previously published osteotomy callus distraction models [4–6]. In sheep metatarsi, axial compression of 0.5 mm led to greater bone formation than compression with 0.18 mm [6]. Similar results are known for fracture healing studies in which greater predominantly compressive IFM led to higher callus density [13, 14]. Moderate compression at 10% strain increased osteogenic activity of bone cells [15]. In contrast to the experiment described herein, endochondral ossification and cartilage formation could be detected in previous callus distraction and fracture healing studies submitted to moderate predominantly compressive stimulation [6, 13, 16]. Although Sox9 positive cells and Collagen Type II could be detected in the current experiment, there were no terminally differentiated chondrocytes or proteoglycan production. Because Runx2 and Sox9 were co-expressed in the same region, Sox9-positve cells were suggested to be osteo-chondroprogenitors as previously described [17].

There are three possible explanations for the pure intramembranous bone formation seen in this experiment as well as for the endochondral bone formation additionally seen in other studies focused on the influence of cyclic compression: differences in direction of IFM, the vascularization in the healing zone, and a possible triggering of osteogenic cell differentiation by tensile strain during distraction may all account for the discrepancies.

Firstly, stimulation of the regenerate tissue after the callus distraction process was solely by controlled cyclic compression in the present model. Tissue deformation due to movements in other directions was avoided by the stiff fixator-system connected to the intact tibia. In osteotomy models previously described [1, 6, 16, 18], other movements, such as bending and shear, could not be completely prevented. Cyclic external loads and muscle forces act within the osteotomy site and the bridging fixator systems cannot be perfectly rigid. Therefore, unquantifiable, auxiliary IFM is present alongside the mechanical motion parameters of interest in osteotomy models. An experiment on different hybrid external fixators reported that shear movement occurs in vitro even under pure axial compression [19] and shear movement generally exceeded axial compression in patients with correction osteotomies [20]. From fracture healing studies it is well known that shear movements are particularly detrimental for the healing process and induce more cartilage formation [21–23]. Low fixator stiffness was identified as one cause of endochondral ossification, and the more oblique tibia loading delayed healing compared to axially loaded radii in DO studies on dogs [1, 24]. López-Pliego et al. [18] have indicated that endochondral ossification during distraction osteogenesis has been reported primarily in small animal studies while intramembranous ossification is dominant in large animal models. They explain this result as a function of fixator stiffness as small animal models typically employ flexible monolateral fixators. In contrast, large animal models allow the technical freedom to develop stiffer and more controllable fixation devices. In the study of López-Pliego et al., 10 adult, female, Merino sheep (the same as those used in the present work) were subjected to bone segment transfer using a stiff fixator and transport protocol of 1 mm per day, once daily for 15 days. Subsequently, the specimens were euthanized at 10 different time points between 2 and 510 days after the completion of distraction. Despite the much larger rate of distraction in comparison to that of the present work the, the authors report that foci of endochondral ossification could only be seen in only 3 of the 10 specimens. When considering that the model of López-Pliego et al. was subject to the imperfect stability associated with osteotomy, it seems likely that the appearance of endochonodral ossification in some specimens was due to variation in interfragmentary motion, exceeding some critical threshold in those specimens. Such variations in interfragmentary motion can easily result from surgical variation, interindividual loading conditions among specimens, as well as manufacturing variation of mating parts for each fixator employed. The model described in the present work avoids such issues by eliminating the osteotomy and therefore interfragmentary motion associated with loading and fixation stiffness. Therefore the prevention of shear movement in the present study may be one of the possible reasons for pure intramembranous bone formation.

A second reason for the appearance of endochondral ossification and cartilage formation in osteotomy models of DO [1, 6, 16] but not in our experiment might be the difference in vascularization of the regenerating zone. Blood supply is a prerequisite for intramembranous bone formation whereas endochondral ossification can take place under hypoxic conditions [25, 26]. Drilling bore holes into the cortical wall of the tibia allowed neovascularization of the regenerating tissue by vessels penetrating from the medullary cavity. This led to a very good vessel density as detected in our histological evaluation. The larger compression amplitude during maturation did not affect vessel density which was on par with values from the animals of the low amplitude compression group. Omitting drill holes in the current model, however, significantly suppressed bone formation [7]. In callus distraction models, differences in blood supply were found when full osteotomy models were compared to more vascularity-preserving techniques such as corticotomies [1]. After corticotomy in dogs, the distraction gap bridged by intramembranous ossification even under fixation exhibiting insufficient stability [27]. Therefore, chondrogenic differentiation may occur in either primarily hypoxic sites and/or under shear movement hindering neovascularization. Frierson et al. associated fibrocartilage development after DO with either wire loosening which likely induces shear movement, or the absence of neovascularization [28].

A third reason may be that the distraction process stimulates pure osseous differentiation in regenerating tissue and this influence persists during the maturation phase. Runx2 up-regulation was found in biopsies of tissue regenerates in human patients [29]. and pure intramembranous bone formation was seen in sheep with solitary lateral callus distraction avoiding IFM during the maturation phase in the predicate experiment [7]. Mesenchymal stem cells show up-regulation of osseous genes under tensile strain whereas compression leads to expression of genes representing cartilaginous differentiation [30]. The strain stimulus for osseous differentiation does not stop once the distraction process has finished but rather persists for a significant period of time. The process of calcification of the young regenerate takes about twice as long as the distraction period [1]. Even without any further mechanical stimuli, intramembranous bone formation persists for at least 50 days after callus distraction [7]. The “Tension-stress” effect may superimpose with the stimulus induced by cyclic compression applied in the current experiment. Thus, osseous differentiation may have suppressed or restricted the stimulus for the differentiation of cells to chondrocytes through cyclic compression [3]. Previously, this could be demonstrated when a limited number of strain stimuli where applied in an eight week fracture healing study stabilized by an external fixator [31]. Eight days of small distractions (two-times 0.5 mm/day) followed by a subsequent shortening to the original size of osteotomy gap led to an improved healing process in comparison to a non-stimulated control group. Although the number of IFM cycles due to weight bearing of the sheep and the elasticity of the fixator system far outweighed the few tensile strain stimulations, there was a pronounced osteogenic effect of the tensile stimuli. More bone formation and less cartilage tissue was observed in the stimulated group in comparison to the control group with constant flexible fixation of the fracture. These studies show that tensile strain applied during the distraction phase, triggers cell differentiation in the osseous direction and can partially suppress differentiation to chondrocytes under moderate cyclic compression. This is supported by the result of this study which shows intensive Runx2 staining of cells in the group with the larger compression amplitude (0.6 mm) and no sign of terminal cartilaginous differentiation.

It could be discussed whether the resection of the periosteum in the callus distraction area reduces the capability of precursor cells to differentiate to chondrocytes because the periosteum is a major source of chondrocytes [32]. However, analysis of bone formation directly next to the callus distraction area (outside the area of interest) where the periosteum was kept intact also showed only intramembranous bone formation. This indicates that the main source of precursor cells and osteoblasts in the callus healing area are coming from the vessels originating from the bone marrow and that the “tension-stress effect” may have superimposed with the possible effect of cartilage formation induced by cyclic compression.

Whether much larger cyclic compression strains will hinder pure intramembranous bone formation needs further investigations.

Conclusion

Pure tensile strain in the callus tissue during the distraction procedure induces a strong osteogenic differentiation of mesenchymal precursor cells. This stimulus persists beyond the distraction phase. The mode of intramembranous bone formation remains unaffected when pure cyclic compression is applied after distraction. In this study, the larger amplitude of cyclic compression led to an increased intramembranous bone formation compared to the small amplitude. So far, this effect has only been demonstrated during endochondral ossification. Whether there is a critical value of compressive strain that hinders intramembranous bone formation cannot be definitively answered and needs further investigation.

From a clinical perspective, callus distraction should be performed using a fixation system which eliminates as much shear movement perpendicular to the bone axis as possible following a vascularity-preserving surgical technique. With regards to axial movements, more IFM than expected is tolerable and stimulating for intramembranous bone formation.


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