CC BY
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Prof. dr hab. Jan Kuryszko1, dr Piotr Kuropka1, dr Stepan Kostiuk 2 ®
1Department of Histology and Embriology, Faculty of Veterinaty Medicine, Agricultural University, Kozuchowska 5, 51-631 Wroclaw. Poland 2Ministry of Agriculture Policy of Ukraine, L NAVM after name Gzyckij, Research Inst. Of animal and birds physiology and ecoimmunology
HEALING OF BONES FRACTURES
Zaklad Histologii i Embriologii Wydzial Medycyny Weterynaryjnej. Akademia Rolnicza, ul. Kozuchowska 5, 51-631 Wroclaw, Polska
The article describes the proces s of fractures healing in animals. It includes descriptions of mechanisms from the beginning of the process including inflammation. osteogenic induction and callus formation up to callus remodeling into the finished bone. It takes into the account the influence of osteosynthesis and medical modes of conduct in the healing process.
Key words: bones - fractures.
The fracture emerges, when upper limit of bone compression is surpassed. It causes beginning of numerous following one after another reactions both whole body and specific local, which ends with moment of recovery by bone its biomechanical properties. In the moment of fracture, activation of cascade of processes, leading to removal of damaged tissues, restitution of blood supply and formation of new bone. Fracture healing is one of the most perfect types of all reparation processes in body and in optimal conditions do not leave scar, and remodeling of damaged bone is sometimes very similar to its original form.
It undergoes in several phases:
l.Inflammation and proliferation phase.
In the moment of fracture interruption of adjacent tissues including blood vessels, nerves and bone marrow. As result blooding occurs, hypoxia and degeneration of tissuses. Vasoactive factors are released from destroyed bone and surrounding tissues (kinines, prostaglandins and other) and growth factors. This process took place few hours after trauma and results in vasodilatation in tissuses. Around facture hemorrhage arouses containing extravasated and migrating neutrophiles, lymphocytes, macrophages, mast cells and platelets. Morphotic elements present in hematoma secrete growth factors which influence growth and proliferation of fibroblasts and pluripotential cells of connective tissue and angiogenesis inducing factors [1, 4,5,6,20,29,34,37].
Inflammatory cytokines involved in fracture repair are believed to play a role in initiating the repair cascade following injury. These cytokines are produced and function immediately after injury for a limited time period. At a mid-stage in healing, some of the inflammatory cytokines are up-regulated, so osteoclastogenesis is stimulated to remove mineralized cartilage, and others are induced at a later stage
® Jan Kuryszko, Piotr Kuropka, Stepan Kostiuk, 2010
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during bone remodeling.
Interleukins-1 and -6 (IL-1 and IL-6) and TNF-alfa have been shown to play a role in initiating the repair cascade. They induce a downstream response to injury by recruiting other inflammatory cells, enhancing extracellular matrix synthesis, and stimulating angiogenesis. They are secreted at the injury site by macrophages, inflammatory and mesenchymal cells
In this early stage, elevated levels of M-CSF, RANKL and OPG but the cytokines that have been associated with bone remodeling, including IL1 and IL-6, are absent during this period (1).
The expression of IL-1 and IL-6 rises again in association with bone remodeling during secondary bone formation, whereas the expression of TNF-alfa rises in association with mineralized cartilage resorption by the end of the endochondral phase of fracture repair. In addition to stimulating osteoclast function, TNF-alfa promotes the recruitment of mesenchymal stem cells and induces apoptosis of hypertrophic chondrocytes during endochondral bone formation. Its absence delays the resorption of mineralized cartilage and, consequently, prevents the formation of new bone.
Release of lyzosomal enzymes from neutrophiles facilitate clot lysis. In the moment of fracture. surrounding tissues contain few only osteoblasts and any chondroblasts. These cells would need 20-100 years to heal fracture in thighbone core [18.19]. To secure full and fast fracture healing num ber of osteoblasts increases thousands times, and this take place under control of numerous growth factors. Cell induction begins very early and ends with appernce of inflammation cells [12]. Factors like BMP (bone morphogenetic protein) and other growth factors (PDGF -platelet derived growth factor, TGF b - transforming growth factor beta) micromovements among broken ends. piezoelectric properties of bone and oxygen partial tension have great influence on bone induction. [35].
Interactions between cells and bone matrix take place. Cells produce and secrete specific polypeptides and especially growth factors. They are chemical regulation factors. If cell synthesizing factor and target celi is that same celi these factors are called as autocrine, when peptide from synthesizing celi re ach target celi by diffusion process - paracrine, when by blood from distant cell - endocrine [18]. Under control of these factors proliferation of osteogenic cell of periosteum, endosteum and bone marrow take place. This proliferation begins shortly after trauma (8 - 12hours), overlapping bloodshot phase. Proliferative reaction is most active place of fracture [18,40]. Its intensivity decreases with distance from place of fracture, however can be observed on whole bone and also other adjacent bones. This is major cause of thickening of periosteum [18, 19]. Mast cells present in clot release heparin, which play important role in angio- and osteogenesis [16,17,30]. Simultaneously removal of destroyed tissues by microphages (neutrophils), and later by macrophages and osteoclasts and release of growth factors from bone matrix. Specific circulation can be observed in place of fracture. In firs days after fracture periosteal circulation is present and subsequently is replaced by endosteal circulation [21]. Since 6 th day after fracture there are both circulation peri- and endosteal. Endosteal circulation and pH changes during healing process. Proper development of circulation is significant for
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beginning and development of healing process [35].
2. Callus formation phase. Proliferation and differentiation of pluripotential cells of endosteum, periosteum and bone marrow causes formation of fibro-cartilagineus and fibroosseous callus [18]. Chondroblasts present in callus produce mitogenic growth factors. which increase synthesis of collagen II type and hialuronic acid what is characteristic for cartilage. Appeared osteoblasts synthesize growth factors which affect synthesis of collagen type 1- 3 characteristic for bone. Accumulated in hematoma macrophages and osteoblasts secrete collagenase- enzyme for collagen fibers lysis. Osteoclasts cell appears to and together with macrophages continue to remove dead remnants of bone. Macrophages secrete also: prostaglandin PGE2, which stimulate synthesis of woven bone and increases speed of bone remodeling [1,27,31]; cytokines IL-1 - induce osteoblasts to secrete factors for osteoclasts activation [1,6, 31,41]; TNF-a, TNF-b increase bone resorbtion [31]; Tgf-b, BMP-2, GDF-10 - induce proliferation of stromal fibroblast osteogenic cells. In this period monocytes in reaction with T- lymphocytes secrete adequate limfokines activating osteoclasts [36]. Space between broken end s is then filled with cells and penetrating blood vessels. Subperiosteal bone synthesis take place in areas adjacent to fracture zone, however in fracture zone chondroblasts appeal replacing fibro-vascular joint by chondroid tissue. Clinically it is expressed by pain, swelIing and movement reduction between broken ends[13].
3. Modeling and remodeling phase may be active for many years. In this phase. removal of necrotic bone take place, and remodeling of newly synthesized woven bone. There are numerous celIular mechanizms and mutual interactions cellsmatrix. The most active osteoclats together with osteoblasts provide bone remodeling. The activity of osteoclasts depends on number and distance of osteoblasts, which guide their action [14, 37, 39]. The highest activity can be observed closely to bone marrow. Resorption lacunas, formed by osteoclasts are filled directly by celI s of marrow origin. Cartilage is replaced by woven bone by the endchondral ossification process. As the result of bone resorbtion during modeling and remodeling phase from bone are released collagenase- resistant glikoprotein (BMP - bone morfogenetic protein), mitogenic and transforming growth factor, osteonectin and osteocalcin. At end of this phase healing process may be considered as finished. Successive replacement of woven bone by lamellar bone. restoration of marrow cave. osteons canal and normai bone size. Ability to bone model ing is limited after reaching by skeletal bones adult age. This is why, in adult, x-ray pictures show some under periosteal thickenings [19].
In 60-s years term primary healing (without callus formation phase) was introduced. This kind of process take place in very stable osteosynthesis and close tights of broken ends where the space between them is not larger then l mm. Significant in this case is systematic osteon remodeling with minimal bone resorption. Such healing with rigid fixation clinically is rarely observed [12]. Until today, the role of nervous system is not fully explained during fracture healing. Some researches [23,28] shows interdependence of peripheral nerves with bone metabolism. In this process calcytonin dependent related peptide (CGRP). vasoactive intestine peptide (VIP), substation P (SP), tyrosine neuropeptide Y (NPY) oraz DbH (dopamine-b
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hydroxylasis) are involved. In all bone types CGRP is present, however SP accompany so called bone-marrow varicoses and external part of periosteum. VIP is present on all layers of periosteum with culmination in layer adjacent to bone. rather locates with single blood vessels. NPY accompany blood vessels present both in bone and periosteum, rarely this neuropeptide is present in endosteal cells. DbH accompanies blood vessels and nerves of periosteum and endosteum.
Perturbations of fracture healing.
For appropriate callus formation, and its subsequent remodeling and modeling appropriate vascularisation is essential. As it was mentioned above, fracture healing runs in paralel and overlaping phases. In microscope, chondrogenesis and woven bone formation in one zone, in other hematoma organisation ore bone remodeling can be visible. It suggests, that different factors may activate and control every phase of fracture healing. Furthermore disturbances of fracture healing may affect single stage only with appropriate run of others. So fracture healing is controlled generally, and some of the factors appeared but which have no significant action in one stage may activate only another stages [23]. Classifications of fracture healing perturbations are based upon X-ray analysis (retarded joint, lack of joint, al!eged joint), rather on biomechanical aspects of fracture healing and on biology on cellular level. Introduced new techniques of fracture treatment pay more attention on bone biology so significantly decrease percentage of joint perturbations. At present, 5% of all treated fractures shows some perturbances [13].
Problems connected with fracture healing can be divided into the 3 groups
[15]:
I. Perturbances of iatrogenic cause, with appropriate biological potential of bone. In this group, biological potential is correct, but because of technique used perturbances make healing process less effective. The cause of these perturbances can be: inappropriate placement, to big distance between broken ends, micromovements, advantage of cutting forces over compressing forces, to close osteosynthesis, bad vascularization of broken ends caused by surgery, infection, delayed surgery . These technical irregularities are in 70-80 % reason of retarded joints or lack of joint out of all fractures. This concern lamellar bone and rather spongious bone. In su ch cases radiograms taken some time after fracture shows appropriate amount of callus with persistent crack between ends. When bone fragments in osteosynthesis are placed to far away from each other then arousing heamatoma is relatively big. Inside haematoma there is low pH and partial oxygen tension what is caused by difficult blood inflow and intake by extravesiculated erythroctes. Thus pluripotential cells differentiate into the chondroblasts but not osteoblasts [2]. This differentiation is supported by overmovements by broken ends. Chondroblasts are spherical in shape and are resistant to pressure and hypoxia. Factors inhibiting angiogenesis are also released. Fibro-cartilagineus callus joins broken ends. Hyaline cartilage fills central part of callus. With elimination of movement between ends and improvement of oxygen tension cartilage is replaced by bone be endochondral ossification with woven bone formation. There is significant retardation in fracture healing. Persistent movement between ends result that the ends are covered by hyaline cartilage what drive directly to formation of alleged joint. Enlarged gap between broken ends
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determines perturbances in celi determination in proliferation phase. Despite osteogenic cells accumulation and growth factors in fracture gap there is no celi activation. Signals are not reaching their target cells. Fibroblast or lipoblasts are fornled in place of chondro or osteoblasts [19]. Space between end is filled by fibrotlS tissue with few cells. Overmovements between ends is not allowing soft tissue for reparation processes and periosteal revascularisation, and causes fibrocartilaginous joint, what is a reason of lack of joint [12]. Moreover, overmovement causes reduction in blood tlow (10 times lower than normal). Properly controlled micromovement causes smali irritants which prolongate intlamation phase of sourrounding tissuses improving angiogenesis. Overstiffed osteosynthesis with gap over 1 mm, result in bone resorption in broken ends, reducing intlamation phase extending heal ing process [12].
Infections are also important factor influencing celi differentiation and model ing phase. Osteoinduction phase is arrested so phagocytes appeared [7]. Accumulated in inflammtion zones monocytes and macrophages release IL-1 and cytotoxic products, which release factors intluencing fibroblasts proliferation. Presence of fibrous tissue instead of bone is visible. Radiologically it manifests by lack of callus with visible gap between broken ends. Delayed surgery discontinue some of healing phases putting back all healing phases to the begining, forcing macroorganizm to activate short-term local factors, because general factors (hormones, drugs, race, sex. age) have permanent influence on bone biology [19].
II. Second group of perturbances is a group of imbalanced biological functions, which causes ineffectiveness of medical treatment. Biological factor can delay or even make impossible bone healing. The are responsible for about 20% of lack of joint [19]. Similar to the first group it touches compact bone. One of these perturbances is inability to callus formation. Callus appears in small amounts or is not present. X-ray pictures taken 2-3 months after fracture do not show signs of healing. This may be because of:
- inappropriate biological processes are unable to their synthesis. because in induction phse instead of bone- fibrous loose connective tissue is formed.
- general metabolic dysfunctions not allow bone mineralization thus it cannot be seen in X-ray examination. Specific scyntygraphic examination shows so called "cold bone". This slows or stops healing process. In som e cases, in gap some of undifferentiated tissue remains in the gap, in other large amount of solid connective tissue causing fibrous artificial joint [19]. These perturbances ma by caused by radiotherapy, cytostatic drugs. local denervations, non-steride anti-inflammatory drugs (indocid- shortening of inflammation phase)[12.19]. Callus formation, beginning of remodeling and modeling processes require activation of precursor cell. This activation leads to appearance of new cell populations. Some of them support blood vessels formation in numerous tissuses [19]. Osteoblasts synthesize and initiate mineraliztion of bone matrix. Osteoclasts play role in model ing and remodeling phase playing main role in substytution of woven bone by secondary bone.
Perturbances of mediator processes may cause slow callus formation and slow replacement by secondary bone. In case of perturbances in blood flow reduced ability to local capilaries network formation remains [35]. In gap hole reduction in oxygen
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presence, and chondroblasts are present. Daleyed bone healing is observed. Some of the illnesses causing angiogenesis inhibition such as: diabetes - causes retardation in long bones. peripherial neuopaties, large local denervations (syryngomielia). tabes dorsalis, cutting of periferial nerves, acute radiation [19], exertional compartment syndrome [12] and others influence perturbances in fracture healing.
Perturbation of callus mineralization show up in osteomalation. rare in rachitis vitamin D dependent. It leads to lack of adhesion. Lack of mineralization of callus inhibits its ability to remodeling, because this process can start when callus is fully mineralized. So minerals can also play important role in activation of mediator processes. Mineralized bone tissue is potent activator of osteoclasts action [38]. If proliferation processes runs correctly, but in place of osteoblasts or chondroblasts fibroblasts are present or lipoblasts, gap between broken ends is filled with fibrous tissue- so alleged joint is formed [19,32]. The cause may be hyperthroidis, neuroma, diabetal neuropathies [19].
Disturbances during remodeling or modeling phase.
Makroorganizm is unable to replace woven bone by secondary bone. Such changes can be observed in osteomalatia and rachitis, and it is caused by lack of ability to mineralization. Unmineralized bone is not removed by osteoclasts, for which mineralized bone is strongest activation signal. [38]. Inability to mineralization is a barrier when bone cannot be properly modeled. Factors that influence modeling process have mechanical nature and usually this is strain acting on elastic bone created by muscles and body weight [12]. There is optimal range for mechanical limb use. Both, to big or to smali loadings may have unfavourabled influence. Osteoblasts by their connection in osteon canal with neighbors osteoblasts and lining bone trabecules cells and bone surface have so called memory of shape [26]. To high loads causes transmission of signals to bone marrow and fast bone formation in place of highest stain and bone resorption in compression zones [26]. If range of mechanical activity is between limit then healing process is accelerated [12].
III. Third group is combination of the former.
Most of healing perturbances reslllt from problems come into being in firs week after frature. It apply biological and technical disturbances.
Alleged joints.
Alleged joint is terminal form of fracture healing perturbations and fixed lack of joint between broken ends [11,32]. This determine persistent interruption of bone and blood vessel continuity and is a certain form of adaptation to conditions present after fracture, In this course secondary mineralogical fractures (radiologically ilustrated) take place [33] and fibrous connection of bone ends. The most often cause is persistent micromovement and infection in fracture gap [22]. X-ray analysis symptomes of alleged joint are: enlarged gap hole, solid bone marrow and sclerotisation of broken ends. There are two types of alleged joints:
1. hypertrophic alleged joint - is characterized by abllndand aillIs on broken ends, similar to elephant foot or horse hoof. A paradox exist, because with periferial vasularization inreased ischemia between broken ends is visible. M^jor causes are: -unproper technique used with proper biological function,
- persistent movement between broken ends - bone fragment dislocation.
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2. atrophic alleged joint - appeal' with large bone loss. fractures with free nonvasularized bone fragment, multiparticle fractures with necrosis of broken ends.
The cause can be both biological as bad technique used and infection. In case of elongation of faction disturbances of celi differentiation, which result is presence of fibroblasts. In infection case, inhibition of osteoinduction take place, so cells differentiate into the monocytes and macrophages with fibrous tissue synthesis in place of bone. Disturbances in molecular signals transmission to osteogenic cells take place.
This division is based upon radiology index, but is not patognomic for pathophysiologic process. More penetrating picture is possibile in endosteal venography. which show lack of venous connection between broken ends. It allows early differentiation between alleged joint and retardation of broken ends adhesion. In alleged joint each end has separated circulation and restoration of endosteal venous sinuses has been never observed if development of callus was stopped in proliferation phase. In retardation of broken ends adhesion connection of venous circulation is restored with presence of central sinus vein, so future adhesion is possible. Microangiogrpaphy of alleged joint show central anemia with increase in periferial vascuIarisation, spindle shaped periosteum indentical on both ends. lt seems that both, vascular and osteogenic healing front were frozen in time and were unable to join broken end in periosteal and endosteal zone. From biological point of view two types of alleged joints can be distinguished:
- alleged joint synovial type okreslany described mainly basing on scyncygraphic analysis. Under light microscope in within limits of structure like joint bursa, synovial membrane and fluid and broken ends covered with hyaline cartilage can be distinguished. This type of pathology is present in fractures placed near joints. and is rare in fractures of corps of long bones.
- alleged joint fibrous type - broken ends are connected by fibrous tissue with few only cells and without blood vessels. Histologically three different types of alleged joints can be distinguished:- without blood vessel, where real avascular pseudoarthrosis can be observed. Space between broken end is filled with dense irregular connective tissue. alleged joint with la ck of circulation. where fibrous tissue contains vessels. These vessels have narrow light and without erythrocytes. There are no necessary signals to activate them. In this picture healing process is inhibited in angio- and osteogenesis phase.
- alleged joint with presence of dystrophic vessels. Vessel light is visible, but erythrocytes are not present.
Summary
1. fracture healing undergoes in three phases: inflammatory and proliferation phase, callus formation phase and modeling and remodeling phase.
a. Inflammation and proliferation phase characterize the presence of processes leading to removal of tissue damage, restoration of blood supply and formation of new bone.
b. New bone formation take place in aftermath of proliferation and differentiation of multipotential cells of perioseum, endosteum and bone marrow.
c. during remodeling an model ing phase replacement of calltls by secondary
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bona take place. 2. Perturbances of fractures healng result from unproper technique used wit h maintained biological potential of biological dysfunction leading to lack of efficiency of medical treatment.
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