Effect of storage and preconditioning of healing rat Achilles tendon on structural and mechanical properties.

Tendon tissue storage and preconditioning are often used in biomechanical experiments and whether this generates alterations in tissue properties is essential to know. The effect of storage and preconditioning on dense connective tissues, like tendons, is fairly understood. However, healing tendons are unlike and contain a loose connective tissue. Therefore, we investigated if storage of healing tendons in the fridge or freezer changed the mechanical properties compared to fresh tendons, using a pull-to-failure or a creep test. Tissue morphology and cell viability were also evaluated. Additionally, two preconditioning levels were tested. Rats underwent Achilles tendon transection and were euthanized 12 days postoperatively. Statistical analyzes were done with one-way ANOVA or Student's t-test. Tissue force and stress were unaltered by storage and preconditioning compared to fresh samples, while high preconditioning increased the stiffness and modulus (p ≤ 0.007). Furthermore, both storage conditions did not modify the viscoelastic properties of the healing tendon, but altered transverse area, gap length, and water content. Cell viability was reduced after freezing. In conclusion, preconditioning on healing tissues can introduce mechanical data bias when having extensive tissue strength diversity. Storage can be used before biomechanical testing if structural properties are measured on the day of testing.

Chronic anterior tibial stress fractures in athletes: No crack but intense remodeling.

PURPOSE: Delayed healing of anterior tibial stress fractures in athletes is related to high tensional forces acting across a putative fracture gap. These forces lead to crack propagation and create strains that exceed tissue differentiation thresholds for new bone to form in the gap. The "dreaded black line" is a radiographic hallmark sign of stress fractures considered to represent a transverse fracture gap. However, whether a fracture gap truly exists at the microscopic level remains unclear. The aim of this study was to describe the area of the "dreaded black line" microscopically and to identify signs of delayed healing. METHODS: Between 2011 and 2016, we included seven athletes with chronic anterior mid-shaft tibial stress fractures. The fracture site was excised as a cylindrical biopsy. The biopsy was evaluated with micro-CT and histology. The formation of new bone in the defect was evaluated radiographically. RESULTS: The "dreaded black line" seen on pre-operative radiographs in all patients could not be seen on the microscopic level. Instead, the area of the putative crack showed widened resorption cavities, lined with active osteoblasts, and surrounded by immature bone. This area of intense remodeling seemed to create a false impression of a fracture line on radiographs. Complete cortical continuity was restored at the biopsy site at median 8 months (range 6-13 months). CONCLUSION: Tibial stress fractures in athletes normally show no fracture defect, but a region of increased remodeling. The healing process is already ongoing but seems mechanically insufficient.

Depletion of cytotoxic (CD8+) T cells impairs implant fixation in rat cancellous bone.

As cytotoxic (CD8+ ) T cells seem to impair shaft fracture healing, we hypothesized that depletion of CD8+ cells would instead improve healing of cancellous bone. Additionally, we also tested if CD8-depletion would influence the healing of ruptured Achilles tendons. Rats received a single injection of either anti-CD8 antibodies or saline and put through surgery 24 h later. Three different surgical interventions were performed as follows: (1) a drill hole in the proximal tibia with microCT (BV/TV) to assess bone formation; (2) a screw in the proximal tibia with mechanical evaluation (pull-out force) to assess fracture healing; (3) Achilles tendon transection with mechanical evaluation (force-at-failure) to assess tendon healing. Furthermore, CD8-depletion was confirmed with flow cytometry on peripheral blood. Flow cytometric analysis confirmed depletion of CD8+ cells (p < 0.001). Contrary to our hypothesis, depletion of CD8+ cells reduced the implant pull-out force by 19% (p < 0.05) and stiffness by 34% (p < 0.01), although the bone formation in the drill holes was the same as in the controls. Tendon healing was unaffected by CD8-depletion. Our results suggest that CD8+ cells have an important part in cancellous bone healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Cyclin-dependent kinase 8/19 inhibition suppresses osteoclastogenesis by downregulating RANK and promotes osteoblast mineralization and cancellous bone healing.

Cyclin-dependent kinase 8 (CDK8) is a mediator complex-associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8-and its paralog CDK19-have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow-derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF-κB) ligand (RANKL)-induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate-resistant acid phosphatase (TRAP) activity and C-terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF-κB, nuclear factor of activated T-cells 1 (NFATc1), dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K in RANKL-stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration.

Abaloparatide versus teriparatide: a head to head comparison of effects on fracture healing in mouse models.

Background and purpose - Teriparatide accelerates fracture healing in animals and probably in man. Abaloparatide is a new drug with similar although not identical effects on the teriparatide receptor. Given at 4 times the teriparatide dose in a human osteoporosis trial, abaloparatide increased bone density more than teriparatide, and both reduced fracture risk. We investigated in mice whether abaloparatide stimulates fracture healing, and if it does so with the suggested dose effect relation (4:1). Patients and methods - In a validated mouse model for metaphyseal healing (burr hole with screw pull-out), 96 mice were randomly allocated to 11 groups: control (saline), teriparatide or abaloparatide, where teriparatide and abaloparatide were given at 5 different doses each. In a femoral shaft osteotomy model, 24 mice were randomly allocated to 3 groups: control (saline), teriparatide (15 µg/kg) or abaloparatide (60 µg/kg). Each treatment was given daily via subcutaneous injections. Results were evaluated by mechanical testing and microCT. Results - In the metaphyseal model, a dose-dependent increase in screw pull-out force could be seen. In a linear regression analysis (r = 0.78) each increase in ln(dose) by 1 (regardless of drug type) was associated with an increase in pull-out force by 1.50 N (SE 0.18) (p < 0.001). Changing drug from teriparatide to abaloparatide increased the force by 1.41 N (SE 0.60; p = 0.02). In the diaphyseal model, the callus density was 23% (SD 10), 38% (SD 10), and 47% (SD 2) for control, for teriparatide and abaloparatide respectively. Both drugs were significantly different from controls (p = 0.001 and p = 0.008), but not from each other. Interpretation - Both drugs improve fracture healing, but in these mouse models, the potency per µg of abaloparatide seems only 2.5 times that of teriparatide, rather than the 4:1 relation chosen in the clinical abaloparatide-teriparatide comparison trial.

Osteoblast precursors and inflammatory cells arrive simultaneously to sites of a trabecular-bone injury.

Background and purpose - Fracture healing in the shaft is usually described as a sequence of events, starting with inflammation, which triggers mesenchymal tissue formation in successive steps. Most clinical fractures engage cancellous bone. We here describe fracture healing in cancellous bone, focusing on the timing of inflammatory and mesenchymal cell type appearance at the site of injury Material and methods - Rats received a proximal tibial drill hole. A subgroup received clodronate-containing liposomes before or after surgery. The tibiae were analyzed with micro-CT and immunohistochemistry 1 to 7 days after injury. Results - Granulocytes (myeloperoxidase) appeared in moderate numbers within the hole at day 1 and then gradually disappeared. Macrophage expression (CD68) was seen on day 1, increased until day 3, and then decreased. Mesenchymal cells (vimentin) had already accumulated in the periphery of the hole on day 1. Mesenchymal cells dominated in the entire lesion on day 3, now producing extracellular matrix. A modest number of preosteoblasts (RUNX2) were seen on day 1 and peaked on day 4. Osteoid was seen on day 4 in the traumatized region, with a distinct border to the uninjured surrounding marrow. Clodronate liposomes given before the injury reduced the volume of bone formation at day 7, but no reduction in macrophage numbers could be detected. Interpretation - The typical sequence of events in shaft fractures was not seen. Mesenchymal cells appeared simultaneously with granulocyte and macrophage arrival. Clodronate liposomes, known to reduce macrophage numbers, seemed to be associated with the delineation of the volume of tissue to be replaced by bone. Most fracture healing studies in animal models concern cortical bone in shafts. However, most fractures in patients occur in cancellous bone in the metaphysis, such as the distal radius or in the vertebrae. A growing body of evidence suggests that there are important differences between the healing processes in cortical and cancellous bone.

Shining dead bone-cause for cautious interpretation of [18F]NaF PET scans.

Background and purpose - [18F]Fluoride ([18F]NaF) PET scan is frequently used for estimation of bone healing rate and extent in cases of bone allografting and fracture healing. Some authors claim that [18F]NaF uptake is a measure of osteoblastic activity, calcium metabolism, or bone turnover. Based on the known affinity of fluoride to hydroxyapatite, we challenged this view. Methods - 10 male rats received crushed, frozen allogeneic cortical bone fragments in a pouch in the abdominal wall on the right side, and hydroxyapatite granules on left side. [18F]NaF was injected intravenously after 7 days. 60 minutes later, the rats were killed and [18F]NaF uptake was visualized in a PET/CT scanner. Specimens were retrieved for micro CT and histology. Results - MicroCT and histology showed no signs of new bone at the implant sites. Still, the implants showed a very high [18F]NaF uptake, on a par with the most actively growing and remodeling sites around the knee joint. Interpretation - [18F]NaF binds with high affinity to dead bone and calcium phosphate materials. Hence, an [18F]NaF PET/CT scan does not allow for sound conclusions about new bone ingrowth into bone allograft, healing activity in long bone shaft fractures with necrotic fragments, or remodeling around calcium phosphate coated prostheses.

Marrow compartment contribution to cortical defect healing.

Background and purpose - Healing of shaft fractures is commonly described as regards external callus. We wanted to clarify the role of the bone marrow compartment in the healing of stable shaft fractures. Patients and methods - A longitudinal furrow was milled along the longitudinal axis of the femoral shaft in mice. The exposed bone marrow under the furrow was scooped out. The mice were then randomized to no further treatment, or to receiving 2 silicone plugs in the medullary canal distal and proximal to the defect. The plugs isolated the remaining marrow from contact with the defect. Results were studied with histology and flow cytometry. Results - Without silicone plugs, the marrow defect was filled with new bone marrow-like tissue by day 5, and new bone was seen already on day 10. The new bone was seen only at the level of the cortical injury, where it seemed to form simultaneously in the entire region of the removed cortex. The new bone seemed not to invade the marrow compartment, and there was a sharp edge between new bone and marrow. The regenerated marrow was similar to uninjured marrow, but contained considerably more cells. In the specimens with plugs, the marrow compartment was either filled with loose scar tissue, or empty, and there was only minimal bone formation, mainly located around the edges of the cortical injury. Interpretation - Marrow regeneration in the defect seemed to be a prerequisite for normal cortical healing. Shaft fracture treatment should perhaps pay more attention to the local bone marrow.

Temporal role of macrophages in cancellous bone healing.

Macrophages are important phagocytosing and cytokine producing cells with effects on fracture healing. We used clodronate-containing liposomes to reduce the number of macrophages, in order to study their role in the early phases of cancellous bone healing. Holes were drilled bilaterally into the cancellous bone of the proximal metaphysis of the tibia of 60 mice. A screw was inserted in the hole in the right tibia. The day of surgery was day 0. Clodronate-containing liposomes were injected intraperitoneally as a single injection either day -4 or -1 (before surgery) or day 1 or 3 (after surgery). A control group underwent surgery as above, but received no clodronate. The mice were killed day 7. The mechanical quality of the new formed cancellous bone holding the screw was evaluated by a pull-out test. The contents of the drill hole in the left tibia was analyzed by microCT. Another set of 20 mice received a drill hole in the metaphysis of the right tibia, and were given either clodronate or saline injections days -3 and -2. The animals were killed day 1 and 3. Flow cytometry was used to analyze the composition of macrophage subpopulations in the regenerating tissue. Flow cytometry showed that clodronate injections day -3 and -2 led to a decrease in mature monocytes day 1 together with an increase in immature monocytes. On day 3 this effect had mostly disappeared, suggesting that the effect of the injections lasted 3 to 5days. Mechanical testing revealed that the injections prior to surgery decreased the strength of the new formed bone, holding the screw, by about half. Bone density in the drill hole was similarly reduced. In contrast, the injections given day 1 and 3 had smaller and statistically insignificant effects. Since their depletion at later time points failed to produce a significant effect, it seems that the role of macrophages in cancellous bone is most crucial during the first two days after trauma.

Isolated metaphyseal injury influences unrelated bones.

Background and purpose - Fracture healing involves different inflammatory cells, some of which are not part of the traditional bone field, such as B-cells and cytotoxic T-cells. We wanted to characterize bone healing by flow cytometry using 15 different inflammatory cell markers in a mouse model of metaphyseal injury, and incidentally discovered a previously unknown general skeletal reaction to trauma. Material and methods - A bent needle was inserted and twisted to traumatize the cancellous bone in the proximal tibia of C57/Bl6 female mice. This is known to induce vivid bone formation locally in the marrow compartment. Cells were harvested from the injured region, the uninjured contralateral tibia, and the humerus. The compositions of the immune cell populations were compared to those in untraumatized control animals. Results - Tibial metaphyseal injury led to substantial changes in the cell populations over time. Unexpectedly, similar changes were also seen in the contralateral tibia and in the humerus, despite the lack of local trauma. Most leukocyte subsets were affected by this generalized reaction. Interpretation - A relatively small degree of injury to the proximal tibia led to systemic changes in the immune cell populations in the marrow of unrelated bones, and probably in the entire skeleton. The few changes that were specific for the injury site appeared to relate to modulatory functions.

Earlier effect of alendronate in mouse metaphyseal versus diaphyseal bone healing.

Healing of injured cancellous bone is characterized by a transient stage of rapid bone formation throughout the traumatized bone volume, often followed by similarly rapid resorption. This is different from the slower diaphyseal healing via an external callus. We, therefore, hypothesized that antiresorptive treatment might have an earlier positive effect in cancellous bone healing than in diaphyseal fractures. One hundred and twenty-three male C57bl6 mice received either an internally stabilized diaphyseal osteotomy of the femur or a screw inserted into the tibial metaphysis. The mice were randomized to daily alendronate injections (200 µg/kg/day), or control injections, and killed for mechanical testing after 14, 21, or 28 days. The hypothesis was tested by a three-way Anova (time, site, and drug). The ultimate force was increased by bisphosphonate treatment in both models. There was a significant interaction between time, site, and drug (p < 0.001) so that the full positive effect of alendronate was evident in the metaphysis at 14 days, but first after 28 days in the diaphysis. While the early effect in the metaphysis might be translated into earlier healing, the late effect in the diaphysis was due to delayed remodeling of the callus, which might have less clinical importance. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:793-799, 2017.

Experimental models for cancellous bone healing in the rat.

BACKGROUND AND PURPOSE: Cancellous bone appears to heal by mechanisms different from shaft fracture healing. There is a paucity of animal models for fractures in cancellous bone, especially with mechanical evaluation. One proposed model consists of a screw in the proximal tibia of rodents, evaluated by pull-out testing. We evaluated this model in rats by comparing it to the healing of empty drill holes, in order to explain its relevance for fracture healing in cancellous bone. To determine the sensitivity to external influences, we also compared the response to drugs that influence bone healing. METHODS: Mechanical fixation of the screws was measured by pull-out test and related to the density of the new bone formed around similar, but radiolucent, PMMA screws. The pull-out force was also related to the bone density in drill holes at various time points, as measured by microCT. RESULTS: The initial bone formation was similar in drill holes and around the screw, and appeared to be reflected by the pull-out force. Both models responded similarly to alendronate or teriparatide (PTH). Later, the models became different as the bone that initially filled the drill hole was resorbed to restore the bone marrow cavity, whereas on the implant surface a thin layer of bone remained, making it change gradually from a trauma-related model to an implant fixation model. INTERPRETATION: The similar initial bone formation in the different models suggests that pull-out testing in the screw model is relevant for assessment of metaphyseal bone healing. The subsequent remodeling would not be of clinical relevance in either model.

Anti-RANKL treatment improves screw fixation in cancellous bone in rats.

Bisphosphonates improve implant fixation in randomised clinical trials of knee prostheses, hip prostheses and dental implants. However, a limited amount of bone resorption is required for bisphosphonates to exert an effect. Anti-RANKL treatment does not have this limitation, and we therefore tested whether if they might be more effective for improvement of implant fixation. This is of interest, as anti-RANKL treatment with denosumab is now in common clinical use. Male SD rats received a stain-less steel screw in the right proximal tibia and a drill hole in the left (n = 42). They were randomised to subcutaneous injections of either alendronate (20 µg/kg/day), alendronate (200 µg/kg/day), osteoprotegerin with an Fc tag (OPG-Fc; 8 mg/kg, twice weekly), or saline control. After 4 weeks, the fixation of the steel screw was measured by pull-out test. The tibia with the drill hole was evaluated with µCT. OPG-Fc increased the pull-out force compared to saline controls by 153% (p < 0.001). There was no significant difference between OPG-Fc and the alendronate groups. OPG-Fc increased the bone density (BV/TV) in the previous drill hole compared to controls 7-fold (p < 0.001). This increase was higher than with any alendronate dose (p < 0.001). OPG-Fc increased the bone density of the L5 vertebral body, but there was no significant difference between OPG-Fc and alendronate. Our results suggest that screw fixation in cancellous bone can be dramatically improved by an anti-RANKL agent. The effect was comparable to very high bisphosphonate doses. Screw insertion in cancellous bone elicits a metaphyseal fracture healing response, and our findings might be relevant not only for implant fixation, but also for fracture healing in cancellous bone.