Theodore Miclau

Does adult fracture repair recapitulate embryonic skeletal formation

Bone formation is a continuous process that begins during fetal development and persists throughout life as a remodeling process. In the event of injury, bones heal by generating new bone rather than scar tissue; thus, it can accurately be described as a regenerative process. To elucidate the extent to which fetal skeletal development and skeletal regeneration are similar, we performed a series of detailed expression analyses using a number of genes that regulate key stages of endochondral ossification. They included genes in the indian hedgehog (ihh) and core binding factor 1 (cbfa1) pathways, and genes associated with extracellular matrix remodeling and vascular invasion including vascular endothelial growth factor (VEGF) and matrix metalloproteinase 13 (mmp13). Our analyses suggested that even at the earliest stages of mesenchymal cell condensation, chondrocyte (ihh, cbfa1 and collagen type II-positive) and perichondrial (gli1 and osteocalcin-positive) cell populations were already specified. As chondrocytes matured, they continued to express cbfa1 and ihh whereas cbfa1, osteocalcin and gli1 persisted in presumptive periosteal cells. Later, VEGF and mmp13 transcripts were abundant in chondrocytes as they underwent hypertrophy and terminal differentiation. Based on these expression patterns and available genetic data, we propose a model where Ihh and Cbfa1, together with Gli1 and Osteocalcin participate in establishing reciprocal signal site of injury. The persistence of cbfa1 and ihh, and their targets osteocalcin and gli1, in the callus suggests comparable processes of chondrocyte maturation and specification of a neo-perichondrium occur following injury. VEGF and mmp13 are expressed during the later stages of healing, coincident with the onset of vascularization of the callus and subsequent ossification. Taken together, these data suggest the genetic mechanisms regulating fetal skeletogenesis also regulate adult skeletal regeneration, and point to important regulators of angiogenesis and ossification in bone regeneration.

Altered fracture repair in the absence of MMP9

The regeneration of adult skeletal tissues requires the timely recruitment of skeletal progenitor cells to an injury site, the differentiation of these cells into bone or cartilage, and the re-establishment of a vascular network to maintain cell viability. Disturbances in any of these cellular events can have a detrimental effect on the process of skeletal repair. Although fracture repair has been compared with fetal skeletal development, the extent to which the reparative process actually recapitulates the fetal program remains uncertain. Here, we provide the first genetic evidence that matrix metalloproteinase 9 (MMP9) regulates crucial events during adult fracture repair. We demonstrate that MMP9 mediates vascular invasion of the hypertrophic cartilage callus, and that Mmp9-/- mice have non-unions and delayed unions of their fractures caused by persistent cartilage at the injury site. This MMP9- dependent delay in skeletal healing is not due to a lack of vascular endothelial growth factor (VEGF) or VEGF receptor expression, but may instead be due to the lack of VEGF bioavailability in the mutant because recombinant VEGF can rescue Mmp9-/- non-unions. We also found that Mmp9-/- mice generate a large cartilage callus even when fractured bones are stabilized, which implicates MMP9 in the regulation of chondrogenic and osteogenic cell differentiation during early stages of repair. In conclusion, the resemblance between Mmp9-/- fetal skeletal defects and those that emerge during Mmp9-/- adult repair offer the strongest evidence to date that similar mechanisms are employed to achieve bone formation, regardless of age.

Minimally invasive plate osteosynthesis and vascularity: preliminary results of a cadaver injection study

A cadaver arterial injection study was performed to analyse the vascular supply to the femur and to study the effects of two surgical plating techniques on femoral vascularity. A 16-hole LC-DCP was applied on the intact femora of five fresh human cadavers. On one side, the plate was inserted using a conventional lateral plate osteosynthesis (CLPO) technique with elevation of the vastus lateralis muscle to expose the shaft. On the contralateral side, the plate was inserted percutaneously beneath the muscle using a minimally invasive plate osteosynthesis (MIPO) technique. After plating, blue silicone dye was injected through the common femoral artery. Cadaveric dissection was then performed to identify the femoral perforating arteries (PAs) and the nutrient artery (NA) of the femur. The pattern of periosteal filling and medullary perfusion of the injected dye was analysed and the topography of the PAs and NA was determined. CLPO placed the PAs and NA of the femur at risk. MIPO maintained the integrity of the PAs and NA and was associated with superior periosteal and medullary perfusion. The results of this study indicate that MIPO is superior to the CLPO in maintaining arterial femoral vascularity and perfusion.

A model for intramembranous ossification during fracture healing.

We have developed a method to study the molecular basis of intramembranous fracture healing. Unlike intramedullary rods that permit rotation of the fractured bone segments, our murine model relies on an external fixation device to provide stabilization. In this study we compare stabilized fracture callus tissues with callus tissues from non‐stabilized fractures during the inflammatory, soft callus, hard callus, and remodeling stages of healing. Histological analyses indicate that stabilized fractures heal with virtually no evidence of cartilage whereas non‐stabilized fractures produce abundant cartilage at the fracture site. Expression patterns of collagen type IIa (colIIa) and osteocalcin (oc) reveal that mesenchymal cells at the fracture site commit to either a chondrogenic or an osteogenic lineage during the earliest stages of healing. The mechanical environment influences this cell fate decision, since mesenchymal cells in a stabilized fracture express oc and fail to express colIIa. Future studies will use this murine model of intramembranous fracture healing to explore, at a molecular level, how the mechanical environment exerts its influence on healing of a fracture.

Variability in the Assessment of Fracture-Healing in Orthopaedic Trauma Studies

BACKGROUND There is a lack of consensus among orthopaedic surgeons in the assessment of fracture-healing. We conducted a systematic review of recent clinical studies of long-bone fracture care that were published in three major orthopaedic journals to identify current definitions of fracture-healing. METHODS MEDLINE and the computerized databases for The Journal of Bone and Joint Surgery (American Volume), The Journal of Bone and Joint Surgery (British Volume), and the Journal of Orthopaedic Trauma were searched from January 1996 through December 2006 with use of title, abstract, keyword, and medical subject headings. Therapeutic clinical studies of long-bone fractures of the appendicular skeleton in adults in which fracture-healing was assessed were selected. Two reviewers independently identified articles and extracted data. Any disagreement was resolved by consensus. We qualitatively and quantitatively summarized the definition of fracture union and the reliability of the assessment of radiographic fracture-healing. RESULTS One hundred and twenty-three studies proved to be eligible. Union was defined on the basis of a combination of clinical and radiographic criteria in 62% of the studies, on the basis of radiographic criteria only in 37%, and on the basis of clinical criteria only in 1%. Twelve different criteria were used to define fracture union clinically, and the most common criterion was the absence of pain or tenderness at the fracture site during weight-bearing. In studies involving the use of plain radiographs, eleven different criteria were used to define fracture union, and the most common criterion was bridging of the fracture site. A quantitative measure of the reliability of the radiographic assessment of fracture union was reported in two studies. CONCLUSIONS We found a lack of consensus with regard to the definition of fracture-healing in the current orthopaedic literature. Without valid and reliable clinical or radiographic measures of union, the interpretation of fracture care studies remains difficult.

The mechanical effect of blocking screws ("Poller screws") in stabilizing tibia fractures with short proximal or distal fragments after insertion of small-diameter intramedullary nails.

OBJECTIVES/HYPOTHESIS To evaluate the mechanical effects of medial and lateral blocking screws in supplementing intramedullary nail fixation of high proximal and low distal tibial fractures treated with small-diameter intramedullary nails. STUDY DESIGN Intact fresh human cadaveric tibiae were sectioned to provide ten distal segments measuring seventy millimeters and ten proximal segments measuring ninety millimeters. In the distal segments, stainless steel solid eight-millimeter tibial nails were advanced to eight millimeters from the ankle joint. Two transverse and one anterior-posterior (AP) locking screw were inserted using a custom-made jig. The same jig was used for the placement of a medial and a lateral blocking screw (BS) in the AP direction, nine millimeters above the superior most interlocking screw and eight millimeters distal to the lower end of the segment. In the proximal segments, two interlocking screws (both static and dynamic screws) were placed in a medial-lateral direction with the use of the insertion handle. A jig was used for placement of a medial and a lateral BS in the AP direction, nine millimeters below the lower transverse interlocking screw and sixteen millimeters proximal to the lower end of the segment. The bone-implant construct (BIC) was embedded and fixed in a materials testing machine. The BICs were loaded in the medial-lateral direction at a distance of 185 millimeters from the nail ends with loads from -150 newtons to + 150 newtons. Force-displacement curves were recorded before and after insertion of the BSs. RESULTS In proximal BICs, the addition of BSs decreased the deformation of the BICs 25 percent, from 8.9 +/- 1.9 degrees [mean +/- standard deviation (SD)] in the control group to 6.8 +/-1.1 degrees in the BS group (mean +/- SD) (p < 0.0001). In distal BICs, the addition of BSs decreased the deformation of the BICs 57 percent, from 9.5 +/- 1.4 degrees (mean +/- SD) in the control group to 4.0 +/- 1.0 degrees in the BS group (mean +/- SD) (p < 0.0001). CONCLUSIONS The study suggests that medial and lateral blocking screws can increase the primary stability of distal and proximal metaphyseal fractures after nailing and can be an effective tool for selected cases that exhibit malalignment and/or instability.

Effect of cefazolin and vancomycin on osteoblasts in vitro.

The effect of cefazolin and vancomycin on osteoblast-like cells was studied. Cells from the MG-63 human osteosarcoma cell line were grown in antibiotic free media and exposed to concentrations of cefazolin and vancomycin at order of magnitude intervals between 0 and 10,000 Ug/ml. For cefazolin, a second interval was performed between 100 and 1000 ug/ml to define toxic levels more accurately. Cell number and 3H-thymidine incorporation at 0, 24, and 72 hours were determined. The results of this study show that local levels of vancomycin of 1000 μg/ml and less have little or no effect on osteoblast replication, and concentrations of 10,000 μg/ml cause cell death. Concentrations of cefazolin of 100 μg/ml and less have little or no effect on osteoblast replication, 200 μg/ml significantly decrease cell replication, and 10,000 μg/ml cause cell death. The authors conclude that vancomycin is less toxic than is cefazolin to osteoblasts at higher concentrations and may be a better antibiotic for local administration in the treatment of similarly sensitive bacterial infections.

Common Molecular Pathways in Skeletal Morphogenesis and Repair

ABSTRACT: The formation of bone is a continual process in vertebrate development, initiated during fetal development and persisting in adulthood in the form of remodeling and repair. The remarkable capacity of skeletal tissues to regenerate has led to the hypothesis that the molecular signaling pathways regulating skeletogenesis are shared during fetal development and adult wound healing. A number of key regulatory pathways that are required for endochondral ossification during fetal development are described, and their reintroduction in fracture repair demonstrated. Secreted proteins such as Sonic and Indian hedgehog exert their effect on pattern formation and chondrogenesis in the appendicular skeleton, partly through regulation of molecules such as bone morphogenic proteins (Bmps) and parathyroid hormone‐related peptide (PTHrP). Once chondrocytes have matured and hypertrophied, they undergo apoptosis and are replaced by bone; the transcription factor Cbfal plays a critical role in this process of chondrocyte differentiation and ossification. Analyses of the expression patterns of these genes during fracture healing strongly suggest that they play equivalent roles in adult wound repair. Knowledge acquired through the study of fetal skeletogenesis will undoubtedly contribute to an understanding of fracture repair, and subsequently guide the development of biologically based therapeutic interventions.

Bone toxicity of locally applied aminoglycosides.

Summary: The effect of tobramycin on osteoblasts was studied. Osteoblast-like cells from the MG-63 human osteosarcoma cell line were grown in antibiotic-free media and exposed to concentrations of tobramycin: first at order of magnitude intervals between 0 and 10,000 μg/ml, and then at closer intervals between 100 and 1,000 μg/ml to more accurately define toxic levels. Cell number and 3H-thymidine incorporation at 0, 24 and 72 h were determined. Similarly, primary cultures of rat osteoblasts were exposed to the same concentrations of tobramycin to confirm the validity of the MG-63 cell line as a model for nontransformed cells. The results of this study demonstrate that local levels of tobramycin of ≤200 μg/ml have little or no effect on osteoblast replication. Concentrations of 400 μg/ml significantly decreased cell replication, and concentrations of 10,000 μg/ml caused cell death. Results obtained with primary rat osteoblast cultures were similar to those obtained from the MG-63 cultures at the tested tobramycin concentrations.

Role of Matrix Metalloproteinase 13 in Both Endochondral and Intramembranous Ossification during Skeletal Regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13−/− mice is intrinsic to cartilage and bone. Mmp13−/− mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.