Fracture vs. LSJL gene expression

Differential gene expression analysis in fracture callus of patients with regular and failed bone healing.

"Between March 2006 and May 2007, a total of 130 patients who were surgically treated at the Berufsgenossenschaftliche Unfallklink Ludwigshafen were screened for the study. Tissue samples of patients with normal and failed fracture healing were collected intraoperatively. Patients were divided into groups depending on the fracture date, and only patients with fractures two to four weeks old and patients with non-unions more than 9 months old were included in the final analysis."

Individuals from 18-80 were used.

"In chondrogenesis, fibronectin is thought to have a significant role in the differentiation of mesenchymal cells to chondral cells."

"The cytoskeletal gene ACTA2, which encodes the human aortic smooth muscle actin, is more than two fold higher expressed in tissue samples of patients with non-unions. Actin is the major component of microfilaments, and plays an important role in maintaining cell shape and movement. Smooth muscle α-actin is expressed in vascular smooth muscle cells and fibroblasts."

Genes upregulated in non-union fractures versus union fractures also upregulated in LSJL:

Acta2

Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

"Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1){up} on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing."

"A majority of bone marrow cells expressed EGR-1 [in the fracture callus], while no EGR-1 was detected in the bone-embedded osteocytes"

"Cartilage formation in EGR-1−/− callus extended frequently perpendicular to the periosteal bone, while cartilage in wt callus formed adjacent to the bone surface"

"The EGR-1−/− mouse callus showed a dumbbell-shaped, enlarged area of ossification"

EGR-1-/- had cartilage deposits within the muscle.

"expression of both PDGF-A/B and fibroblast growth factor 2 is positively regulated by EGR-1"

The biology of fracture healing.

Minor motion is good for fracture healing whereas too much delays fracture healing.

"Immediately following the trauma, a hematoma is generated and consists of cells from both peripheral and intramedullary blood, as well as bone marrow cells. The injury initiates an inflammatory response which is necessary for the healing to progress{microfracture may not induce an inflammatory response thus can it induce the healing process?}. The response causes the hematoma to coagulate in between and around the fracture ends, and within the medulla forming a template for callus formation."

"The initial proinflammatory response involves secretion of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6{up}, IL-11 and IL-18."

"The TNF-α concentration has been shown to peak at 24h and to return to baseline within 72h post trauma."

"TNFR1 is always expressed in bone whereas TNFR2 is only expressed following injury"

"IL-1 and IL-6 are believed to be most important for fracture healing"<-so the upregulation of IL-6 may be sufficient.

"TGF-β2, -β3 and GDF-5 are involved in chondrogenesis and endochondral ossification, whereas BMP-5 and -6 have been suggested to induce cell proliferation in intramembranous ossification at periosteal sites."

"Bone remodelling has been shown to be a result of production of electrical polarity created when pressure is applied in a crystalline environment. This is achieved when axial loading of long bones occur, creating one electropositive convex surface, and one electronegative concave surface, activating osteoclastic and osteoblastic activity respectively. By these actions the external callus is gradually replaced by a lamellar bone structure, whereas the internal callus remodelling re-establishes a medullar cavity characteristic of a diaphyseal bone."

"Bone on one side of the cortex must unite with bone on the other side of the cortex to re-establish mechanical continuity. If the gap between bone ends is less than 0.01 mm and interfragmentary strain is less than 2%, the fracture unite by so-called contact healing. Under these conditions, cutting cones are formed at the ends of the osteons closest to the fracture site. The tips of the cutting cones consist of osteoclasts which cross the fracture line, generating longitudinal cavities at a rate of 50–100 μm/day. These cavities are later filled by bone produced by osteoblasts residing at the rear of the cutting cone. This results in the simultaneous generation of a bony union and the restoration of Haversian systems formed in an axial direction. The re-established Haversian systems allow for penetration of blood vessels carrying osteoblastic precursors. The bridging osteons later mature by direct remodelling into lamellar bone resulting in fracture healing without the formation of periosteal callus."

"Gap healing differs from contact healing in that bony union and Haversian remodelling do not occur simultaneously. It occurs if stable conditions and an anatomical reduction are achieved, although the gap must be less than 800 μm to 1 mm. In this process the fracture site is primarily filled by lamellar bone oriented perpendicular to the long axis, requiring a secondary osteonal reconstruction unlike the process of contact healing. The primary bone structure is then gradually replaced by longitudinal revascularized osteons carrying osteoprogenitor cells which differentiate into osteoblasts and produce lamellar bone on each surface of the gap. This lamellar bone, however, is laid down perpendicular to the long axis and is mechanically weak. This initial process takes approximately 3 and 8 weeks, after which a secondary remodelling resembling the contact healing cascade with cutting cones takes place. Although not as extensive as endochondral remodelling, this phase is necessary in order to fully restore the anatomical and biomechanical properties of the bone"

"The acute inflammatory response peaks within the first 24h and is complete after 7 days"

Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing.

"Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4(th) and 7(th) day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th)-7(th) days after fracture. Sclerostin expression diminished during the first days after fracture.
The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing."

Genetic factors responsible for long bone fractures non-union.

"We carried out studies in patients with delayed long bone fractures estimating the frequency of mutation of genes crucial for pathogen recognition (TLR2, TLR4 and CD14), and elimination (CRP, IL-6, IL-1ra), as well as wound healing (TGF-β). The molecular milieu regulating healing process (IGF-1, COLL1a, TGF-β, BMP-2, and PDGF) was validated by Western blot analysis of the gap tissue.
Microbiological investigations showed the presence of viable bacterial strains in 34 out of 108 gaps in patients with non-healing fractures (31.5%) and in 20 out of 122 patients with uneventful healing (16.4%). The occurrence of mutated TLR4 1/W but not 2/W gene was significantly higher in the non-healing infected than sterile group. In the non-healing infected group 1/W mutated gene frequency was also higher than in healing infected. In the TGF-β codon 10 a significantly higher frequency of mutated homozygote T and heterozygote C/T in the non-healing infected versus non-healing sterile subgroup was observed. Similar difference was observed in the non-healing infected versus healing infected subgroup. The CRP (G1059C), IL1ra (genotype 2/2), IL-6 (G176C), CD14 (G-159T), TLR2 (G2259A) and TLR4/2 (Thr399Ile) polymorphisms did not play evident role in the delay of fracture healing.
Individuals bearing the mutant TLR 4 gene 1/W (Asp299Gly) and TGF-β gene codon 10 mutant T and T/C allele may predispose to impaired pathogen recognition and elimination, leading to prolonged pathogen existence in the fracture gaps and healing delays."