Louis C. Gerstenfeld

Fracture healing as a post‐natal developmental process: Molecular, spatial, and temporal aspects of its regulation

Fracture healing is a specialized post‐natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing, these processes take place in a post‐natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the embryonic differentiation and growth of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (1) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular differentiation during repair; (4) the role of three key groups of soluble factors, pro‐inflammatory cytokines, the TGF‐β superfamily, and angiogenic factors, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture. J. Cell. Biochem. 88: 873–884, 2003.

Differential Temporal Expression of Members of the Transforming Growth Factor β Superfamily During Murine Fracture Healing

Fracture healing is a unique postnatal repair process in which the events of endochondral and intramembranous bone formation follow a definable temporal sequence. The temporal patterns of messenger RNA (mRNA) expression for members of the transforming growth factor β (TGF‐β) superfamily were examined over a 28‐day period of fracture healing in mouse tibias. Bone morphogenetic protein 2 (BMP‐2) and growth and differentiation factor 8 (GDF8) showed maximal expression on day 1 after fracture, suggesting their roles as early response genes in the cascade of healing events. Restricted expression of GDF8 to day 1, in light of its known actions as a negative regulator of skeletal muscle growth, suggests that it may similarly regulate cell differentiation early in the fracture healing process. GDF5, TGF‐β2, and TGF‐β3 showed maximal expression on day 7, when type II collagen expression peaked during cartilage formation. In contrast, BMP‐3, BMP‐4, BMP‐7, and BMP‐8 showed a restricted period of expression from day 14 through day 21, when the resorption of calcified cartilage and osteoblastic recruitment were most active. TGF‐β1, BMP‐5 and BMP‐6, and GDF10 were constitutively expressed from day 3 to day 21. However, during the same time period, GDF3, GDF6, and GDF9 could not be detected, and GDF1 was expressed at extremely low levels. These findings suggest that several members of the TGF‐β superfamily are actively involved in fracture healing and although they are closely related both structurally and functionally, each has a distinct temporal expression pattern and potentially unique role in fracture healing.

Molecular Mechanisms Controlling Bone Formation during Fracture Healing and Distraction Osteogenesis

Fracture healing and distraction osteogenesis have important applications in orthopedic, maxillofacial, and periodontal treatment. In this review, the cellular and molecular mechanisms that regulate fracture repair are contrasted with bone regeneration that occurs during distraction osteogenesis. While both processes have many common features, unique differences are observed in the temporal appearance and expression of specific molecular factors that regulate each. The relative importance of inflammatory cytokines in normal and diabetic healing, the transforming growth factor beta superfamily of bone morphogenetic mediators, and the process of angiogenesis are discussed as they relate to bone repair. A complete summary of biological activities and functions of various bioactive factors may be found at COPE (Cytokines & Cells Online Pathfinder Encyclopedia), http://www.copewithcytokines.de/cope.cgi.

Expression of differentiated function by mineralizing cultures of chicken osteoblasts

This report documents osteoblast differentiation in vitro, as demonstrated by the 50-100X increase of proteins which are known markers of the osteoblast phenotype. Collagen type I and osteocalcin synthesis and accumulation, alkaline phosphatase activity, and matrix calcification show similar temporal relationships that are analogous to those seen during in vivo bone development. Chicken embryonic osteoblast progenitor cells were selected by initial growth at low densities in minimal medium. Upon subcultivation into nutrient-enriched medium at higher cell densities, near homogeneous populations of osteoblasts were obtained as demonstrated by the greater than 80% enrichment of cells positive for alkaline phosphatase activity. A comparison was made between cells grown in the presence or absence of 10 mM beta-glycerolphosphate (beta-GPO4), a chemical stimulant of matrix calcification, as a function of time. Cultures treated with beta-GPO4 showed visible calcification at Day 12 when culture monolayers became confluent. By Day 30, numerous large foci of calcification were visible and a 20-fold increase in calcium (Ca) content was observed. In contrast, untreated cultures had only a 3-fold increase in Ca content with many smaller diffuse areas of calcification. DNA, RNA, and total protein levels were nearly identical between the two cultures, indicating that beta-GPO4 had no marked effect on either cell proliferation or transcriptional activity. The major collagen type produced by either culture was type I, with no detectable type III as determined by CNBr peptide mapping and delayed reduction analysis. Alkaline phosphatase activity showed a rapid approximately 50-fold induction by Day 18 and remained elevated in control cultures. However, cultures treated with beta-GPO4 demonstrated a rapid 80% decline of enzyme activity after 18 days. In contrast, total osteocalcin levels showed a 100-fold induction by Day 18 and remained elevated in both control and beta-GPO4-treated cultures throughout the time period examined. While the overall levels of osteocalcin were the same in beta-GPO4-treated and untreated cultures, 2- to 5-fold more osteocalcin was associated with the more mineralized matrices of the beta-GPO4-treated cultures. In order to confirm the association of osteocalcin with areas of mineralization, co-localization of mineral to osteocalcin and collagen was carried out by combining vital labeling with tetracycline and immunofluorescent staining with anti-osteocalcin and anti-collagen antibodies. Both collagen and osteocalcin showed strong localization with areas of mineralization.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing.

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony‐stimulating factor [M‐CSF], and osteoprotegerin ligand [receptor activator of NF‐κB ligand (RANKL)]), as well as inflammation (tumor necrosis factor α [TNF‐α] and its receptors, and interleukin‐1α [IL‐1α] and ‐β and their receptors) were analyzed over a 28‐day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M‐CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF‐α, lymphotoxin‐β (LT‐β), IL‐1α, and IL‐1β showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF‐α receptors (p55 and p75) and the IL‐1RII receptor showed identical patterns of expression to their ligands, while the IL‐1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF‐α and IL‐1α expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF‐α expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M‐CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF‐α and IL‐1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.

Impaired Fracture Healing in the Absence of TNF‐α Signaling: The Role of TNF‐α in Endochondral Cartilage Resorption

TNF‐α is a major inflammatory factor that is induced in response to injury, and it contributes to the normal regulatory processes of bone resorption. The role of TNF‐α during fracture healing was examined in wild‐type and TNF‐α receptor (p55−/−/p75−/−)‐deficient mice. The results show that TNF‐α plays an important regulatory role in postnatal endochondral bone formation.

Differential inhibition of fracture healing by non‐selective and cyclooxygenase‐2 selective non‐steroidal anti‐inflammatory drugs

Non‐steroidal anti‐inflammatory drugs (NSAIDs) specifically inhibit cyclooxygenase (COX) activity and are widely used as anti‐arthritics, post‐surgical analgesics, and for the relief of acute musculoskeletal pain. Recent studies suggest that non‐specific NSAIDs, which inhibit both COX‐1 and COX‐2 isoforms, delay bone healing. The objectives of this study were 2‐fold; first, to measure the relative changes in the normal expression of COX‐1 and COX‐2 mRNAs over a 42 day period of fracture healing and second, to compare the effects of a commonly used non‐specific NSAID, ketorolac, with a COX‐2 specific NSAID, Parecoxib (a pro‐drug of valdecoxib), on this process. Simple, closed, transverse fractures were generated in femora of male Sprague‐Dawley rats weighing approximately 450 g each. Total RNA was prepared from the calluses obtained prior to fracture and at 1, 3, 5, 7, 10, 14, 21, 35 and 42 days post‐fracture and levels of COX‐1 and COX‐2 mRNA were measured using real time PCR. While the relative levels of COX‐1 mRNA remained constant over a 21‐day period, COX‐2 mRNA levels showed peak expression during the first 14 days of healing and returned to basal levels by day 21. Mechanical properties of the calluses were then assessed at 21 and 35 days post‐fracture in untreated animals and animals treated with either ketorolac or high or low dose parecoxib. At both 21 and 35 days after fracture, calluses in the group treated with the ketorolac showed a significant reduction in mechanical strength and stiffness when compared with controls (p < 0.05). At the 21‐day time point, calluses of the parecoxib treated animals showed a lower mean mechanical strength than controls, but the inhibition was not statistically significant. Based on physical analysis of the bones, 3 of 12 (25%) of the ketorolac‐treated and 1 of 12 (8%) of the high dose parecoxib‐treated animals showed failure to unite their fractures by 21 days, while all fractures in both groups showed union by 35 days. Histological analysis at 21 days showed that the calluses in the ketorolac‐treated group contained substantial amounts of residual cartilage while neither the control nor the parecoxib‐treated animals showed comparable amounts of cartilage at this stage. These results demonstrate that ketorolac and parecoxib delay fracture healing in this model, but in this study daily administration of ketorolac, a non‐selective COX inhibitor had a greater affect on this process. They further demonstrate that a COX‐2 selective NSAID, such as parecoxib (valdecoxib), has only a small effect on delaying fracture healing even at doses that are known to fully inhibit prostaglandin production.

Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34).

BACKGROUNDnRecombinant human parathyroid hormone (PTH [1-34]; teriparatide) is a new treatment for postmenopausal osteoporosis that can be systemically administered for the primary purpose of increasing bone formation. Because several studies have described the enhancement of fracture-healing and osteointegration in animals after use of PTH, we sought to critically analyze this skeletal effect.nnnMETHODSnTwo hundred and seventy male Sprague-Dawley rats underwent standard, closed femoral fractures and were divided into three groups that were administered daily subcutaneous injections of 5 or 30 mug/kg of PTH (1-34) or vehicle (control). The dosing was administered for up to thirty-five days. Groups were further divided into three subgroups and were killed on day 21, 35, or 84 after the fracture. The bones were subjected to mechanical torsion testing, histomorphometric analysis, or microquantitative computed tomography.nnnRESULTSnBy day 21, calluses from the group treated with 30 mug of PTH showed significant increases over the controls with respect to torsional strength, stiffness, bone mineral content, bone mineral density, and cartilage volume. By day 35, both groups treated with PTH showed significant increases in bone mineral content and density and total osseous tissue volume, and they demonstrated significant decreases in void space and cartilage volume (p < 0.05). Torsional strength was significantly increased at this time-point in the group treated with 30 mug of PTH (p < 0.05). While dosing was discontinued on day 35, analyses performed after eighty-four days in the group treated with 30 mug of PTH showed sustained increases over the controls with respect to torsional strength and bone mineral density. No change was noted in osteoclast density at the time-points measured, suggesting that treatment with PTH enhanced bone formation but did not induce bone resorption.nnnCONCLUSIONSnThese data show that daily systemic administration of PTH (1-34) enhances fracture-healing by increasing bone mineral content and density and strength, and it produces a sustained anabolic effect throughout the remodeling phase of fracture-healing.

Expression of bone‐specific genes by hypertrophic chondrocytes: Implications of the complex functions of the hypertrophic chondrocyte during endochondral bone development

Endochondral bone formation is one of the most extensively examined developmental sequences within vertebrates. This process involves the coordinated temporal/spatial differentiation of three separate tissues (cartilage, bone, and the vasculature) into a variety of complex structures. The differentiation of chondrocytes during this process is characterized by a progressive morphological change associated with the eventual hypertrophy of these cells. These cellular morphological changes are coordinated with proliferation, a columnar orientation of the cells, and the expression of unique phenotypic properties including type X collagen, high levels of bone, liver, and kidney alkaline phosphatase, and mineralization of the cartilage matrix. Several studies indicate that hypertrophic chondrocytes also express osteocalcin, osteopontin, and bone sialoprotein, three proteins which until very recently were widely believed to be restricted in their expression to osteoblasts. Recent studies suggest that the hypertrophic chondrocytes are regulated by the calcitropic hormones, morphogenic steroids, and local tissue factors. These considerations are based on the regulation by 1,25(OH)2D3 and retinoids of the cartilage specific genes as well as osteopontin and osteocalcin expression in hypertrophic chondrocytes. They are also based on the effects on growth plate development caused by 1) transgenic ablation of autocrine/paracrine regulators such as PTHrP and of the transcriptional regulator c‐fos and 2) naturally occurring genetic mutations of the FGF receptor. These studies further suggest that specific transcriptional factors mediate exogenous regulatory signals in a coordinated manner with the development of bone. While it has been widely demonstrated that the majority of hypertrophic chondrocytes undergo apoptosis during terminal stages of the developmental sequence, their response to specific exogenous regulatory signals and their expression of bone‐specific proteins give rise to questions about whether all growth chondrocytes have the same developmental fates and have identical functions. Furthermore, specific questions arise as to whether there are similar mechanisms of regulation for commonly expressed genes found in both cartilage and bone or whether these genes have unique regulatory mechanisms in these different tissues. These recent findings suggest that hypertrophic chondrocytes are functionally coupled during endochondral bone formation to the recruitment of osteoblasts, vascular cells, and osteoclasts.

BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP‐7 and BMP‐2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP‐7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP‐7 increased the expression of Sox9, Msx 2, and c‐fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP‐7 treatment and remained elevated throughout the time‐course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP‐7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP‐7‐regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor‐β (TGF‐β), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP‐4, growth and differentiation factor‐1 (GDF‐1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP‐4, and GDF‐1 expression were inhibited by BMP‐7 treatment in a dose‐dependent manner while Wnt 5b and Shh were selectively induced by BMP‐7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP‐7 treatment, however, the period of maximal expression was during the later time‐points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP‐7 activity could be further enhanced twofold by growing the cells in a more nutrient‐rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP‐7 and enhanced by a nutrient‐rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.