Search

BACKGROUND: Substantial evidence exists to show the positive effects of radialextracorporeal shock wave therapy (ESWT) on bone formation. However, it is unknown whether rESWT can act locally at the growth plate level to stimulate linear bone growth. One way to achieve this is to stimulate chondrogenesis in the growth plate without depending on circulating systemic growth factors. We wished to see whether rESWT would stimulate metatarsal rat growth plates in the absence of vascularity and associated systemic growth factors. QUESTIONS/PURPOSES: To study the direct effects of rESWT on growth plate chondrogenesis, we asked: (1) Does rESWT stimulate longitudinal bone growth of ex vivo cultured bones? (2) Does rESWT cause any morphological changes in the growth plate? (3) Does rESWT locally activate proteins specific to growth plate chondrogenesis? METHODS: Metatarsal bones from rat fetuses were untreated (controls: n = 15) or exposed to a single application of rESWT at a low dose (500 impulses, 5 Hz, 90 mJ; n = 15), mid-dose (500 impulses, 5 Hz, 120 mJ; n = 14) or high dose (500 impulses, 10 Hz, 180 mJ; n = 34) and cultured for 14 days. Bone lengths were measured on Days 0, 4, 7, and 14. After 14 days of culturing, growth plate morphology was assessed with a histomorphometric analysis in which hypertrophic cell size (> 7 µm) and hypertrophic zone height were measured (n = 6 bones each). Immunostaining for specific regulatory proteins involved in chondrogenesis and corresponding staining were quantitated digitally by a single observer using the automated threshold method in ImageJ software (n = 6 bones per group). A p value < 0.05 indicated a significant difference. RESULTS: The bone length in the high-dose rESWT group was increased compared with that in untreated controls (4.46 mm ± 0.75 mm; 95% confidence interval, 3.28-3.71 and control: 3.50 mm ± 0.38 mm; 95% CI, 4.19-4.72; p = 0.01). Mechanistic studies of the growth plate’s cartilage revealed that high-dose rESWT increased the number of proliferative chondrocytes compared with untreated control bones (1363 ± 393 immunopositive cells per bone and 500 ± 413 immunopositive cells per bone, respectively; p = 0.04) and increased the diameter of hypertrophic chondrocytes (18 ± 3 µm and 13 ± 3 µm, respectively; p < 0.001). This was accompanied by activation of insulin-like growth factor-1 (1015 ± 322 immunopositive cells per bone and 270 ± 121 immunopositive cells per bone, respectively; p = 0.043) and nuclear factor-kappa beta signaling (1029 ± 262 immunopositive cells per bone and 350 ± 60 immunopositive cells per bone, respectively; p = 0.01) and increased levels of the anti-apoptotic proteins B-cell lymphoma 2 (718 ± 86 immunopositive cells per bone and 35 ± 11 immunopositive cells per bone, respectively; p < 0.001) and B-cell lymphoma-extra-large (107 ± 7 immunopositive cells per bone and 34 ± 6 immunopositive cells per bone, respectively; p < 0.001). CONCLUSION: In a model of cultured fetal rat metatarsals, rESWT increased longitudinal bone growth by locally inducing chondrogenesis. To verify whether rESWT can also stimulate bone growth in the presence of systemic circulatory factors, further studies are needed. CLINICAL RELEVANCE: This preclinical proof-of-concept study shows that high-dose rESWT can stimulate longitudinal bone growth and growth plate chondrogenesis in cultured fetal rat metatarsal bones. A confirmatory in vivo study in skeletally immature animals must be performed before any clinical studies.

Background Osteoarthritis (OA) is one of the leading causes of disability in the world. Several genes are associated with the development of OA, and previous studies have shown adult children of individuals with OA have higher areal bone mineral density (BMD). Because childhood is an important period of growth and bone development, and body composition is known to be associated with BMD, we speculated that there may be differences in growth and bone measures among young children with a genetic predisposition to OA. Questions/purposes (1) Do differences exist at baseline in anthropometric and peripheral quantitative CT (pQCT) measurements between children and grandchildren of individuals with OA and controls? (2) Do children and grandchildren of individuals with OA accrue bone longitudinally at a different rate than controls? Methods Longitudinal anthropometric (height, weight) and bone (cortical and trabecular volumetric BMD and cross-sectional area) measurements by pQCT were obtained at baseline and 18 and 36 months on children (n = 178) and grandchildren (n = 230) of 23 individuals with hip or knee arthroplasty resulting from OA and 23 sex-matched controls (16 females each). Grandchildren (age, 8–30 years) were further categorized as growing (premenarcheal or male < 14 years, n = 99) or mature (≥ 2 years postmenarchal or males ≥ 18 years, n = 96). The remaining 35 grandchildren could not be categorized and were excluded. Results Mature granddaughters and grandsons of individuals with OA had greater trabecular volumetric BMD than controls (236 ± 24 and 222 ± 26 mg/cm3, respectively, for granddaughters, difference of 14 [95% confidence interval {CI}, 1-28] mg/cm3, p = 0.041 and 270 ± 22 and 248 ± 30 mg/cm3, respectively, for grandsons, difference of 22 [95% CI, 1-42] mg/cm3, p = 0.040). Greater trabecular volumetric BMD was observed in daughters of individuals with OA compared with daughters of controls (228 ± 28 and 212 ± 33 mg/cm3, respectively, difference of 18 [95% CI, 3-30] mg/cm3, respectively [p = 0.021]). Growing granddaughters and grandsons of controls had greater decreases in cortical volumetric BMD than grandchildren of individuals with OA (time-by-group [T*G] based on mixed model [± standard error] -9.7 ± 4.3 versus -0.8 ± 4.4 mg/cm3/year, respectively, for granddaughters, difference of 9.0 [95% CI, 2.4-15.5] mg/cm3/year, p = 0.007 and -6.8 ± 3.3 versus 4.5 ± 3.4 mg/cm3/year, respectively, for grandsons, difference of 11.3 [95% CI, 4.3-18.3] mg/cm3/year, p = 0.002). Cortical volumetric BMD was maintained in sons of individuals with OA, but decreased in sons of controls (-0.0 ± 1.5 versus -4.3 ± 1.0 mg/cm3/year, respectively, difference of 4.3 [95% CI, 0.7-7.8] mg/cm3/year, p = 0.019 [T*G]). There was a greater apparent decrease in cross-sectional area among daughters of individuals with OA than in controls (-4.6 ± 0.9 versus -1.7 ± 0.9 mm2/year, respectively, difference of -2.9 [95% CI, -5.3 to -0.6] mm2/year, p = 0.015 [T*G]). Conclusions Several anthropometric and bone differences exist between children and grandchildren of individuals with OA and controls. If these differences are confirmed in additional studies, it would be important to identify the mechanism so that preventive measures could be developed and implemented to slow or reduce OA development. Clinical Relevance Differences in growth and bone development may lead to increased loads on cartilage that may predispose offspring to the development of OA. If these differences are confirmed in additional studies, it would be important to identify the mechanism so that preventive measures could be developed and implemented to slow or reduce OA development.

Skeletal maturity assessment provides information on a child's physical development and expectations based on chronological age. Given recently recognized trends for earlier maturity in a variety of systems, most notably puberty, examination of sex-specific secular trends in skeletal maturation is important. For the orthopaedist, recent trends and changes in developmental timing can affect clinical management (eg, treatment timing) if they are currently based on outdated sources.(1) Has the male or female pediatric skeleton experienced a secular trend for earlier maturation over the past 80 years? (2) Do all indicators of maturity trend in the same direction (earlier versus later)?In this retrospective study, a total of 1240 children were examined longitudinally through hand-wrist radiographs for skeletal maturity based on the Fels method. All subjects participate in the Fels Longitudinal Study based in Ohio and were born between 1930 and 1964 for the "early" cohort and between 1965 and 2001 for the "recent" cohort. Sex-specific secular trends were estimated for (1) mean relative skeletal maturity through linear mixed models; and (2) median age of maturation for individual maturity indicators through logistic regression and generalized estimating equations.Overall relative skeletal maturity was significantly advanced in the recent cohort (maximum difference of 5 months at age 13 years for girls, 4 months at age 15 years for boys). For individual maturity indicators, the direction and magnitude of secular trends varied by indicator type and sex. The following statistically significant secular trends were found: (1) earlier maturation of indicators of fusion in both sexes (4 months for girls, 3 months for boys); (2) later maturation of indicators of projection in long bones in both sexes (3 months for girls, 2 months for boys); (3) earlier maturation of indicators of density (4 months) and projection (3 months) in carpals and density in long bones (6 months), for girls only; and (4) later maturation of indicators of long bone shape (3 months) for boys only.A secular trend has occurred in the tempo of maturation of individual components of the pediatric skeleton, and it has occurred in a sex-specific manner. The mosaic nature of this trend, with both earlier and later maturation of individual components of the skeletal age phenotype, calls for greater attention to specific aspects of maturation in addition to the overall skeletal age estimate. The Fels method is currently the most robust method for capturing these components, and future work by our group will deliver an updated, user-friendly version of the Fels assessment tool.Appreciation of sex-specific secular changes in maturation is important for clinical management, including treatment timing, of orthopaedic patients, because children today exhibit a different pattern of maturation than children on whom original maturity assessments were based (including Fels and Greulich-Pyle).

Radiotherapy used in the treatment of bone and soft tissue sarcomas in pediatric patients often results in undesirable growth plate damage. Radioprotectants may hold promise in the selective protection of growth plate tissue in this setting. In an animal model, the hypothesis tested was that pentoxifylline, selenium, or misoprostol, used in combination with amifostine, would significantly reduce longitudinal growth loss during one radiation dose exposure to a greater extent than the protection provided by only amifostine without increased morbidity or mortality or adverse effects on bone mineral density. Amifostine alone and in combination with each of the other radioprotectants resulted in limb discrepancy reduction to levels significantly less than radiated controls. The tibial length discrepancy in the selenium and amifostine group was 12.1 +/- 0.8%, less than the 15.5 +/- 2.6% tibial length discrepancy in the animals treated with amifostine alone, and less than the mean 18.8% tibial length discrepancy in the radiated limbs without radioprotection. There were no adverse effects on bone density in any group, but the selenium and amifostine group showed some increased mortality. Combinations of amifostine with these radioprotectants show efficacy in growth plate radioprotection and therefore warrant additional study in a clinically relevant fractionated model.

One of the major dilemmas in limb preservation in skeletally immature children involves the ability to maintain leg length equality. Many attempts have been made to design a prosthesis that could be expanded easily either nonoperatively or through a minor surgical procedure. Most of these designs have had mechanical difficulty or the lengthening procedure becomes a major surgical endeavor. The Phenix technology has been used in France for several years. The basic principle involves storage of energy in a spring which is maintained compressed by an original locking system. Once implanted, prosthetic lengthening is achieved via exposure to an external electromagnetic field that pilots the locking system and allows controlled release of the spring energy. Seven Phenix prostheses have been implanted in six patients. All patients had been treated for Stage II-B osteosarcoma. Six of the seven prostheses were implanted during revision procedures in salvage situations; one prosthesis was implanted during an index procedure. The surgical procedures were completed without complications. One patient sustained a fracture of the prosthesis in a fall and had an infection develop after implantation of the second prosthesis. Twenty-one expansions have been performed in six patients (mean lengthening at each procedure, 8 mm). There were no acute complications attributable to the lengthening procedure. Prosthetic expansions required an average of 20 to 30 seconds and were accompanied by very mild discomfort, if any. Most patients were given an oral analgesic either before or during the lengthening procedure. The Phenix prosthesis shows promise in handling the difficult problem of limb preservation in a growing child. Additional investigation is underway regarding limb lengthening and other dynamic applications.

As shown in previous studies, the transforming growth factor beta superfamily of growth factors is involved in many aspects of skeletal development and regulation, including fracture repair and bone regeneration. Several studies have shown transforming growth factor beta messenger ribonucleicacid and protein expression in cells comprising fracture callus. In healing fractures in a chick model, differential isoform expression of the transforming growth factor betas was observed by in situ hybridization, with more prominent expression of the transforming growth factor beta 2 and transforming growth factor beta 3 isoforms. Small amounts of transforming growth factor beta 1 were present in early callus and increased in expression later during chondrogenesis and endochondral ossification. These findings resemble those reported in rat and human fracture callus. Transforming growth factor beta 4 expression was not significant in the chick fracture model. Transforming growth factor beta can function as a morphogen when injected subperiosteally, inducing cartilage and bone formation that morphologically resembles many of the events occurring in fracture callus. Exogenous transforming growth factor beta has been used in several critical size defect models of bone regeneration and fracture healing, with most of the studies showing increased bone or callus formation and increased mechanical stability. Numerous variables, including markedly different dose ranges and differing isoforms, dosing regimens, delivery methods, animal models, and various times and endpoint measures for analysis, make it difficult to comparatively assess the effects of transforming growth factor beta on bone healing. Additional study is necessary to satisfactorily determine the role of transforming growth factor beta in normal fracture healing and its potential for use in augmenting this process.

Bilateral distal radius fractures are rare injuries and only a handful of case reports exist. Understanding the demographic variables and associated injuries in patients with these fractures may improve awareness and treatment of concomitant injuries. We determined the differences in mode of trauma and associated injuries between skeletally mature and skeletally immature patients with bilateral distal radius fractures. We retrospectively reviewed the records of 93 patients with bilateral distal radius fractures. We compared demographic data, fracture patterns, mode of injury, treatment modality, and associated injuries for skeletally mature and immature patients. The mean age of all patients sustaining a bilateral injury was 22.5 years and 61 (71%) were male. Of the 51 (55%) skeletally immature patients, 37 (73%) were male, and 44 (86%) sustained a low-energy mechanism of injury. Of the 42 (45%) skeletally mature patients, 29 (69%) were male, and 37 (88%) sustained a high-energy mechanism of injury. Skeletally mature patients had a 38% rate of associated injuries versus 4% found in skeletally immature patients. Skeletally mature patients sustained bilateral distal radius fractures through higher-energy mechanisms and presented with more frequent associated injuries compared with the skeletally immature patients.

A 24-year-old woman presented with an 11-year history of bilateral hip pain. Radiographs of the hips revealed severe bilateral slipped upper femoral epiphyses; the left side was more severely slipped than the right. While moving the hips under fluoroscopy we observed motion at the physes and reproduced the patient’s pain; the motion confirmed the diagnosis of chronic slipped capital femoral epiphysis. Endocrinology tests showed hypothyroidism. After 1 year of thyroxin therapy, the patient’s pain subsided and radiographs of the hips showed fusion of the physes. This case emphasizes the importance of screening for an endocrine disorder in patients with slipped capital femoral epiphysis particularly in adults and shows fusion can occur once the underlying endocrine abnormality is treated.

A major concern in using growth factors in impaction grafting is the potential stimulation of the osteoclastic lineage. A solution would be using an osteoconductive material resistant to resorption and providing initial stability after reconstruction. Growth factors may promote bone formation in combination with such graft materials. We determined whether OP-1 would promote the incorporation of impacted morselized allografts and tricalcium phosphate/hydroxyapatite (TCP/HA) into host bone, whether bone formation would be preceded by an initial process of accelerated resorption, and whether the response to OP-1 remodeling/incorporation would be dose-related. We performed two bone chamber studies in goats to ascertain the early effects of OP-1 dose on resorption and incorporation of impacted morselized allografts and TCP/HA. After 4 weeks, the incorporation process of impacted morselized allografts and TCP/HA was not promoted by OP-1. We observed no signs of accelerated resorption preceding bone formation. An increase in OP-1 dose resulted in an inhibition of fibrous tissue formation but OP-1 did not promote bone formation. Early failures in impaction grafting, using mixes with OP-1, might be explained by the lack of fibrous tissue ingrowth and not by increased resorption and remodeling.

Injuries to the spine in very young children are comparatively rare. The prevalence of upper cervical injuries and spinal cord injuries is greater. Spinal cord injury is more common in young children and fracture is less common than in older children and adolescents. This is because of the anatomic and biomechanical differences in the growing spine including a more horizontal facet orientation, greater elasticity of the soft tissues, less muscular development, and relatively greater head size compared to the trunk. These features are more pronounced in the very young child. The clinical and radiographic evaluation of small children can be difficult. Unossified bone and physeal cartilage can be confused with fractures. The evaluation, safe transportation, and spinal clearance of the unconscious multiply injured child suspected of having spinal injury present special challenges.

Pediatric fractures are commonly classified into five types: plastic deformation, buckle fracture, greenstick fracture, complete fracture, and physeal injuries. The most important anatomic characteristic in the pediatric skeleton is the presence of growth plates and the thick periosteum. It is important to emphasize that just as adult intra-articular fractures require anatomic reduction, so do pediatric articular injuries. The periosteum in children contributes immensely to rapid fracture healing and helps in the reduction and in the maintenance of reduction. In this study, I review some basic principles for the treatment of fractures in children, the main complications of pediatric fractures, and the outcomes assessment in children. The main complications of fractures in children are malalignment, physeal arrest, and refracture cause by fast fracture healing. Nearly all fractures in children can be treated in a cast without worry about stiff joints or need for physical therapy to mobilize injured joints.

A deeper understanding of skeletal development comes from knowledge of the molecular components, cell and tissue structure, and biophysical and vascular mechanisms underlying physiologic function. Endochondral and intramembranous bone formation mechanisms are active during long bone synthesis. Most endochondral growth in length occurs at the physes by the coordinated actions of cell proliferation, matrix synthesis, and chondrocyte hypertrophy. Several intrinsic molecules act to modulate physeal structure and function whereas extrinsic molecules, such as hormones, provide systemic regulation of growth. Biophysical forces develop intrinsically within the growing bone, serving to enlarge it in three dimensions, while extrinsic forces resist and channel expansion into normal recognizable and functional forms. Newer imaging modalities, such as magnetic resonance imaging, are highly effective in assessing epiphyseal vascularity and differentiating fibrous, cartilaginous, mineralized and osseous tissues in prenatal and postnatal developing bone. This study illustrates the interplay between the molecular and biophysical aspects of normal bone growth, shows ways in which altered development leads to abnormal structures, and provides examples where biologic and biophysical interventions can affect bone development in favorable ways.

We conducted this study to determine whether the small-diameter, press-fit stem of a novel, noninvasive expandable endoprosthetic device implanted in the limbs of 6 pediatric patients with Stage IIB osteosarcoma affected the growth of the physis through which the stem was inserted. Local control of the tumor necessitated resection of the involved growth plate, and implantation of the device required penetration of the adjacent uninvolved bone, through the growth plate, by the stem of the device. We measured longitudinal growth and the rate of growth of the adjacent uninvolved bone in the salvaged limb and of the equivalent bone in the unoperated contralateral limb. In all cases but one (in which the patient's growth in the contralateral limb appeared complete), longitudinal growth continued in the limb into which the device was implanted: the adjacent uninvolved bone in the salvaged limb grew by an average of 2.4 cm, and the equivalent bone in the unoperated contralateral limb grew by an average of 2.3 cm. We conclude that implantation of a smooth, press-fit stem through the central portion of the uninvolved adjacent physis does not result in growth retardation or arrest.

Osteoinductive molecules are characterized by their ability to promote the formation of bone. Most osteoinductive molecules are cytokines, which are extracellular proteins or peptides that mediate cell to cell signaling. Examples of osteoinductive cytokines are certain bone morphogenetic proteins and some growth and differentiation factors. Some osteoinductive molecules are not secreted molecules. LIM mineralization protein-1 is an example of an intracellular osteoinductive molecule. Significant advances have been made in characterizing the molecular composition and mechanism of action of these osteoinductive molecules. Preclinical studies with these molecules have provided better understanding of the doses, formulation, and delivery mechanism necessary for effective bone formation in model systems of spinal fusion and other orthopaedic problems. The current authors will review the most important basic science and preclinical studies involving these osteoinductive molecules.

Tissue engineering can be defined as any effort to create or induce the formation of a specific tissue in a specific location through the selection and manipulation of cells, matrices, and biologic stimuli. The biologic concepts and the biochemical and biophysical principles on which these efforts are based have become an exciting and rapidly evolving field of biomedical research. More importantly, tissue engineering is becoming a clinical reality in the practice of orthopaedic surgery, providing patients and physicians with an expanding set of practical tools for effective therapy. New and improved matrices and bioactive factors inevitably will play important roles in the evolution of orthopaedic tissue engineering. However, tissue engineering never can stray far from fundamental biologic principles, and one of these is that cells do all the work. No new tissue forms except through the activity of living cells. No bone graft, no matrix, no growth factor, no cytokine can contribute to the generation or integration of new tissue, except through the influence it has on the behavior of cells. The efficacy of all current clinical tools depends entirely on the cells in the grafted site, particularly the small subset of stem cells and progenitor cells that are capable of generating new tissue. The current authors review a series of key biologic concepts related to the rational design and selection of composites of cells and matrices in contemporary bone grafting and tissue engineering efforts. The functional paradigms of stem cell biology are reviewed, including self renewal, asymmetric and symmetric mitosis, and lineage restriction. Several potential sources for autogenous stem cells for connective tissues are discussed. Finally, a simple mathematical model is introduced as a tool for understanding the functional demands placed on stem cells and progenitors in a graft site and to provide a conceptual framework for the rational design of cell matrix composite grafts.

This current study describes how human genetic approaches are used to identify and understand the roles of genes and their protein products, during skeletal development, growth, and homeostasis. Searches for the genes responsible for brachydactyly, symphalangism, spondyloepiphyseal dysplasia, and camptodactyly-arthropathy syndrome are presented to exemplify these approaches. The important role of the orthopaedic surgeon in this process is discussed.

The morphologic features of bone bark, a structure surrounding the distal and proximal ends of long bones, were studied in the distal femur, proximal tibia, and proximal fibula of 77 spontaneously aborted human fetuses varying in gestational age from 10 to 20 weeks. Standard histologic techniques used in addition to in situ immunohistochemical staining allowed the examination of the structure of the bone bark and localization of Types 1, 2, and 3 collagens at different gestational ages. The bone bark was shaped like a cylindrical sheath of bone lamellae of varying thickness. The epiphyseal end of the bone bark, known as the groove of Ranvier, was covered outwardly by a fibrous layer and inwardly by the epiphyseal cartilage and contained mesenchymal cells, chondroblastic precursor cells, and densely packed cells differentiating into osteoblasts. Neither the cell density in the groove nor the thickness of the bone bark were identical circumferentially, indicating an unequal growth in width. In addition, the presence of periosteal apposition and endosteal resorption of the bone bark on one side and of endosteal bone deposition accompanied by periosteal resorption of the bone bark on the opposite side support the concept of a spatial drift of bones. These observations furnish histologic proof that groove and bone bark, although assuring an equal growth in length, contribute to an unequal and eccentric growth in width.

Aseptic loosening was identified as the predominant cause of implant related failure in a retrospective study of a consecutive series of 168 Stanmore custom made extendible endoprosthetic replacements used in skeletally immature patients. Most of the replacements were used in the treatment of bone tumor and the remainder for the revision of failed massive endoprosthetic replacements. Since the first Stanmore extendible endoprosthesis was inserted in 1976, 4 types of extension mechanisms have been used. Thirty eight of the 164 cases with followup data were revised, of which 19 were as a result of aseptic loosening. Survival analysis revealed that the overall probability of surviving an implant related failure was 0.512 (+/- 0.005) at 5 years, highlighting the high complication rate of these extendible replacements that required a revision procedure. Sixteen of the 19 aseptic loosening cases were distal femoral replacements. The probability of a patient with a distal femoral replacement surviving aseptic loosening was 0.773 (+/- 0.008) at 5 years. Other modes of implant related failure included jamming of the extending mechanism, infection, and maximum extension of the replacement before skeletal maturity had been reached.

Demineralized matrix was delipidized with chloroform methanol before and after demineralization and implanted in muscle of allogeneic rats. Because lipids are difficult to separate completely from bone collagen, another preparation was gelatinized and delipidized with either chloroform methanol or acetone or both. Bone matrix demineralized without delipidization induced formation of a spherical-shaped deposit of new bone, which was remodeled to form a shell of cortical bone and central pool of normal hematopoietic bone marrow. When the bone was delipidized, only 20% to 25% was resorbed and replaced by new bone; unresorbed matrix failed to recalcify. Gelatinized bone matrix delipidized before implantation was even less well resorbed or replaced by new bone, but 12% to 44% of the matrix residue recalcified. The new deposits of bone were colonized by blood-borne bone marrow-derived stem cells and developed central pools of normal hematopoietic bone marrow. Recalcified residual matrix did not develop bone marrow. Additional investigations are required to determine whether the host bed adipocytes provide the phospholipids for recalcification of bone matrix. There was no preliminary recalcification in matrix-induced bone development, even though the 2 processes may occur simultaneously under specified experimental and pathologic conditions. Additional investigations are in progress to determine whether certain acetone soluble lipids may form the endogenous delivery system for bone morphogenetic protein and induced bone development.

Bone marrow regeneration after injury is preceded by local and systemic osteogenic reactions. Recently, an osteogenic growth peptide was characterized in the regenerating marrow. The osteogenic growth peptide also is abundant in normal serum where it is markedly and transiently increased after marrow injury. This increase and the osteogenic growth peptide-induced stimulation of bone formation in vivo suggest a role for this peptide in mediating the systemic osteogenic response. In vitro, the osteogenic growth peptide is an autocrine mitogen for osteoblastic and fibroblastic cells. It also stimulates alkaline phosphatase activity and matrix mineralization. The serum osteogenic growth peptide is downregulated in osteoporotic ovariectomized mice. The osteogenic growth peptide levels as well as the bone loss, tetracycline uptake, and serum osteocalcin are reversed by exogenously administered osteogenic growth peptide. In normal mice, the osteogenic growth peptide increases white blood cell counts and total femoral bone marrow cellularity. These increases include all the hemopoietic lineages. When given to mice for 1 week before ablative radiotherapy and bone marrow transplantation, synthetic osteogenic growth peptide stimulates the bone marrow transplant engraftment; optimal osteogenic growth peptide doses doubled the survival rate. These data indicate that osteogenic growth peptide has an important role in the pathogenesis and treatment of systemic bone loss and provide a basis for further development of an antiosteoporotic osteogenic growth peptide therapy. It is suggested also that the osteogenic growth peptide promotes hemopoiesis secondary to the stimulation of the stromal (particularly osseous) microenvironment.

Although bone formation traditionally has been difficult to study in vitro, recent improvements in cell culture techniques have made it possible to study primary cultures of fetal rat calvarial osteoblasts, and to examine specific gene events associated with osteoblast proliferation, differentiation, and mineralization of extracellular matrix to form bone nodules. Results of studies show that extracellular matrix proteins such as Type I collagen and osteocalcin are expressed. Also expressed are growth regulatory factors such as the bone morphogenetic proteins, which are presumably secreted and stored in the bone matrix or involved in additional osteoblast differentiation in an autocrine/paracrine role. The bone matrix is 1 source of these growth regulatory factors for bone formation; another is those animal and human tumors associated with osteoblastic metastases. In some of these tumors, an extended form of basic fibroblast growth factor has been identified, and various bone morphogenetic proteins have been found in others.

Prostaglandins are likely to play an important role in the physiologic and pathologic responses of skeletal tissue. They are potent agonists that can stimulate and inhibit bone resorption and formation. In vivo, the major effect of exogenous prostaglandins, particularly prostaglandin E2, is to stimulate resorption and formation. These effects appear to involve replication and differentiation of osteoclast and osteoblast precursors, and to be mediated at least in part by cyclic 3' 5' adenosine monophosphate. Prostaglandins can inhibit the activity of isolated osteoclasts, probably also by a cyclic 3' 5' adenosine monophosphate-mediated mechanism. Inhibition of collagen synthesis can be seen in cell and organ cultures and appears to be caused by a receptor selective for prostaglandins of the F series and to involve activation of protein kinase C. Prostaglandin production by bone cells is regulated highly by mechanical forces, cytokines, growth factors, and systemic hormones. Prostaglandins also can amplify their own production. Regulation is associated with marked changes in the newly described "inducible" prostaglandin G/H synthase with less effect on the constitutive enzyme. Prostaglandins also may play a role in postmenopausal bone loss because estrogen deficiency, which increases bone turnover, can increase prostaglandin production in bone.

Saos-2 cultured human osteosarcoma cells contain an extractable bone inducing agent that can induce heterotopic bone in the muscle of Nu/Nu mice. A semipurified GuHCl extract of Saos-2 cells also can promote healing and complete bony union in otherwise non-healing surgically induced defects of rat femur. Northern blot analyses indicate expression of mRNAs for bone morphogenetic proteins (BMP)-1, 2, 3, 4, 6 and transforming growth factor beta (TGF beta) in Saos-2 cells, and BMP-2, 3, 4, 5, 7 and TGF beta in nonosteoinductive U20S human osteosarcoma cells. Saos-2 cells exceeded U20S cells in expression levels of BMP-1, 3, 4 and TGF beta, whereas U20S cells expressed higher levels of BMP-2, 6 and also expressed trace amounts of BMP-5 and 7 not seen in Saos-2 cells. The authors hypothesize that Saos-2 cells contain an optimal admixture of known bone growth factors plus possible other unknown components that, acting alone or in combination with bone morphogenetic protein and/or TGF beta, can induce bone. Although bone inducing agent-induced heterotopic bones have half lives of only a few weeks, the reparative bone induced by bone inducing agent in femoral defects gives every indication of being permanent and self-sustaining. This suggests a fundamental difference between heterotopic and orthotopic osteoprogenitor cells with those involved in orthotopic bone repair more closely resembling the committed or determined osteoprogenitor cells of marrow as described by Friedenstein.

Only 3 cases of persistent olecranon physes in adults have been reported previously in literature. In each case, physeal nonunion was present bilaterally and either unilateral, bipartite, or tripartite patellae were present. A case of painful unilateral physeal nonunion in an adult weight lifter is presented. The patient had begun competitive weight lifting when he was 14 years old. Radiographs of the contralateral olecranon and patellae were normal. After failure of conservative treatment, surgical union was achieved, using tension band wiring augmented with autologous iliac crest bone graft. The author proposes that unilateral olecranon physeal nonunion has a mechanical etiology as opposed to a genetic etiology in individuals with bilateral physeal persistence. Surgical treatment in the former group is best delayed until after age 19, to allow adequate time for spontaneous union.

The tibiae of rabbits were lengthened by 10 mm in 2 weeks, then approximately one half of the limbs were shortened by 2 mm in 3 days, and left for 3 days. Histological and cytomorphometrical findings of the calluses in both groups were compared. In the shortened callus, marked histologic changes were observed in the central mesenchymal cell layer where the number of the osteoblasts increased 4 fold, and massive primitive fiber bone formation took place. Cartilaginous tissue did not seem to respond to the axial shortening stress.

The authors compared the effects of decreased loading, increased loading, and baseline loading on the early (six weeks) and intermediate (12 weeks) healing of tibial fractures treated with an external fixator in adult dogs. The different loading conditions were verified by gait studies. Periosteal bone in the increased-loading fracture site was significantly increased compared with the decreased-loading site at both time intervals, and with the baseline-loading site at six weeks. The mechanical variables of energy absorption and angle of rotation at six weeks were significantly increased in the increased-loading fracture site compared with the decreased-loading site and baseline-loading site. Blood flow to the increased-loading fracture site was significantly higher than that to the decreased-loading fracture site at six weeks. Five dogs were studied at 12 weeks to compare the effects of decreased loading to increased loading. The increased-loading fracture showed significantly increased rotation, torque, and energy absorption to failure. Blood flow was not measured in the 12-week animals.

Bovine bone morphogenetic protein and associated noncollagenous proteins (bBMP/NCP) were implanted in skull trephine defects, 14 and 22 mm in diameter, in adult minipigs. The defects were larger than the critical size for repair of cranial bones in pigs observed for a period of six to 16 weeks. The trephines were placed either in the nasal bone at eye level, or in the frontal bone. In minipigs the repair was more advanced in trephines in the frontal bone than in the nasal region, but was incomplete in either bone bed. As in dogs, implants of bBMP/NCP in heterotopic sites were resorbed before a competent cell population could respond. Serum anti-BMP antibody was elevated. The IgG levels of the antibody titer showed a clearcut dose-response; the level was significantly higher in one pig with implants of 85 mg of bBMP/NCP proteins in a frontal bone trephine.

Seventy-five fibular specimens were obtained from postmortem donors using aseptic surgical techniques. All specimens were swabbed following the same technique as routinely used for retrieved musculoskeletal grafts. After swabbing, the specimens were placed in BHI-culture medium. Three different protocols were subsequently followed: (1) culture of the entire bone specimen in BHI-culture medium, (2) culture of the swab incubated on blood agar and chocolate plates, and (3) culture of the swab in BHI-culture medium. A control group included 20 sterilized bone specimens that were cultured entirely in BHI-culture medium according to Protocol 1. The sensitivity and negative predictive value were found to be 10% and 9% in Protocol 2 and 13% in Protocol 3. These findings imply that swab cultures are inadequate to detect bacterial contamination of musculoskeletal allografts in all cases. However, the instances of infection after transplantation of allografts bacteriologically screened according to Protocols 1 and 2 do not exceed those reported in other similar series. This suggests an acceptable bioburden.

Chinese hamster ovary (CHO) cells were transfected with the complementary DNA (cDNA) for bone morphogenetic protein-4 (BMP-4) that was derived from the poly A-RNA of a murine (Dunn) osteosarcoma. A clonal line of the transfected, BMP-4 gene expressing cells was expanded and inoculated into the hindlimbs of nude mice to produce BMP-4 secreting tumors. The aims of this study were to investigate the systemic effects of endogenous BMP-4 production on skeletal growth and radiologic density, the local effects of BMP-4 on bone at the tumor-bone interface, and the changes in tumor histology as a result of transfecting CHO cells with the gene for BMP-4. In control mice, mock vector-transfected CHO cells were inoculated in the same manner. Three weeks after the mice received the transfected cells, neither enhanced skeletal growth nor a change in bone density was noted. However, at the site where the bone was in contact with the tumor, new cartilage and bone were consistently observed. The murine BMP-4 transfected CHO tumor contained spicules of new bone that have been described as a histologic feature characteristic of osteosarcoma.

The traditional in vitro cell culture methods provide bone cells on matrix-coated Petri dishes to grow the cells in a monolayer. This limits the usefulness in situations where there is a need to suspend anchorage dependent cells. To promote the massive production of the cultured cells and to enable the suspension of the cells in a medium, microcarrier cell culture was developed and evaluated. Isolated bone cells from rat calvaria were incubated in a microcarrier culture flask with Cytodex 1. The microcarrier cell morphology was examined by a phase contrast microscope, a scanning electron microscope, and a transmission electron microscope. Cell growth, enzyme markers, and biosynthetic characteristics were examined and compared for microcarrier and monolayer methods. The results indicate that all the characteristics of the bone cells, whether cultured in the Petri dish or microcarrier, were the same. Therefore, the studies of the function and behavior of bone cells still remain useful using the microcarrier system culture.

Bone formation by distraction was studied using three different experimental models: (1) Physeal distraction of the sheep radius was performed in 20 animals. (2) Distraction after osteotomy of the radius was carried out in 39 sheep. (3) Mandibular distraction after osteotomy was performed in 17 sheep. Formation of the organic matrix and osteogenesis were studied by radiographic, histologic, and biochemical methods as well as by electron microscopy. The mode of osteogenesis was essentially similar in all of these distraction models. Bone formation was preceded by organization of the collagenous matrix in the distraction area. In the beginning of the distraction, the gap was composed of a heterogeneous cell population, with large polymorphic fibroblast-like cells. The cells in the central part differentiated into fibroblasts, which remained functionally active as long as distraction proceeded. During physeal distraction, bone formed from the epiphyseal and metaphyseal sides as well as from the surrounding perichondrium. Also, in osteotomy distraction of both tubular bone and mandible, bone formed centripetally from the osteotomized bone ends toward the center of the gap. The organic matrix was composed almost solely of Type I collagen in the earliest stages, suggesting that the mode of osteogenesis differs from bone repair by fracture callus. The structure of the distracted segment was mainly lamellar trabecular. Corticalization of the lengthened bone segment occurred gradually after several months.

Noncollagenous proteins, including bone morphogenetic protein (BMP), were extracted in 4 mol/l guanidinium hydrochloride (GuHCl) from the pulverized and HCl-demineralized matrix of reindeer, bovine, sheep and porcine bone. To remove water-soluble material, the GuHCl solution was dialyzed against water and water-insoluble material and redissolved in 4 mol/l GuHCl. Gelatin peptides were removed by extraction in 0.25 mol/l citrate buffer (pH 3.1). The yield consisted mostly of large complexes and protein molecules of molecular weight less than 35,000 daltons. Isoelectric focusing of the material showed three to four different protein molecules: three acidic and one neutral. Bone-forming activity was investigated by implanting 0.6-15.0 mg of partially purified protein preparation into the thigh muscles of BALB mice. Radiologically detectable formation of new bone required 0.6 mg of reindeer BMP, 2.5 mg of bovine BMP, 5.1 mg of sheep BMP, and 8.0 mg of porcine BMP. A rough estimate of the area of the deposits showed that reindeer BMP had the highest bone formation activity, and porcine had the lowest. The formation of new bone was confirmed histologically. It is suggested that the differences in osteogenic activity are due to quantitative differences in BMP constituents or in the degree of complex formation in the protein preparations. Also immune and other defense mechanisms may generate differences in osteogenic response.

Muscle- and synovium-derived mesenchymal-type cells differentiated into cartilage in response to a coating of partially purified rabbit bone morphogenetic protein (BMP) on a substratum of plastic. Observed by means of phase contrast microscopy, light microscope histochemistry, and electron microscopy, the sequence of events on a BMP-coated plastic substratum is the same as that observed on a substratum of bone matrix. The plastic eliminates the possibility of any endogenous allogeneic matrix-derived BMP contaminating the system. The differentiation of cartilage from synoviocytes and subsynovial cells resembles the pathologic process occurring in human chondromatosis.

The effect of functional activity on bone formation and interstitial fluid space in a standardized tibial defect was measured in 19 dogs with one hind limb elevated and the other weight bearing and in 15 dogs bearing weight on all four legs. The interstitial fluid space (V(isf)) of the woven bone of the weight-bearing defect was larger than that in the elevated legs at seven and 14 days. By 28 days, this difference disappeared. In a third group of six dogs, medullary canal pressure was measured in the immobilized and weight-bearing tibias. The pressures were higher in the weight-bearing tibiae at two, three, four, and five days. Because the increase in V(isf) in the weight-bearing defects coincided with the onset of woven bone formation in the defects and an increase in medullary canal pressure, an increase in venous pressure could increase capillary pressure and capillary filtration. Such an increase in filtration might cause the larger V(isf) in the weight-bearing defects and better capillary perfusion of precursor cells of the osteoblast. A hypothesis is that increased fluid flow from increased capillary filtration might produce streaming potentials, a possible signal for increased cellular activity.

By limb lengthening with the Ilizarov method on 24 children with an average age of 11 years, the average lengthening was 50 mm. The results were judged by the healing index and the corticotomy index. The average time for lengthening was 50 days. The healing index was on average 35 days per centimeter. Five complications were noticed during the first stage of the surgical procedure. During the lengthenings, eight cases of joint stiffness were observed, and in five normal biomechanical axes of the limb were modified. Only one abnormal biomechanical axis was perfectly corrected of eight present before surgery. There were three wire-tract infections. Only 13 of 26 patients were without any complications. The Ilizarov method had a lower rate of complication than the Wagner technique, however. Open corticotomy, as modified by De Bastiani, may accomplish lengthenings with somewhat fewer complications.

A comparative analysis was made of subchondral replacement with polymethylmethacrylate and autogeneic bone grafts in defects in the medial femoral condyles of dogs. The defect produced a 50% reduction in subchondral stiffness. An in vitro preparation helped establish that subchondral stiffness returned to normal after reconstruction with polymethylmethacrylate. The in vivo model demonstrated a reduction in subchondral stiffness in both groups at three weeks, but the bone grafted side returned to normal and the methylmethacrylate side recovered to 79% of the control at 12 weeks. There were no deleterious effects on the articular cartilage in either group when analyzed histologically and biochemically. A marked increase in new bone formation and subchondral porosity was found in the polymethylmethacrylate groups. This study supports the clinical use of subchondral polymethylmethacrylate after the exteriorization and curettage of benign bone tumors such as giant cell tumors.

The effects of the immunosuppressive drug cyclosporin A (CsA) on bone induction by demineralized allogeneic (rat) bone matrix (DABM) and demineralized xenogeneic (rabbit) bone matrix (DXBM) were studied. Growing rats were implanted with three samples each of DABM and DXBM. Groups of eight rats were treated with 0.5 or 2 mg CsA/kg body weight for four weeks and compared with a placebo group. Cyclosporin A treatment enhanced bone induction in DABM implants by 40 to 50% at four weeks, whereas there was no difference from the control group at eight weeks. Demineralized xenogeneic bone matrix induced virtually no bone in control rats at four weeks, whereas the net bone formation increased four to five times in both groups of CsA-treated rats. At eight weeks, DXBM without CsA had induced some bone formation, and the amount was almost equal to that of DABM implants in CsA-treated groups. Also, the mineral accretion rates of DXBM were equal to DABM implants in CsA-treated rats. Cyclosporin A treatment doubled the uptake of 45Ca in the orthotopic skeleton (femora) at four weeks without affecting the mineral content, indicating an increased mineral turnover. Immunologic reactions may inhibit bone induction by DXBM, which can be counteracted by treatment with CsA.

The mechanical properties of canine tibias lengthened by the Ilizarov method as well as the degree of healing--as determined by the osseous appearance of the limbs using three imaging modalities--were examined as a function of fixator application time. Sixteen mongrel dogs had right tibial lengthening using a two-ring Ilizarov external fixator. The average lengthening was 27 mm. Four groups of four animals were killed at four, six, eight, and ten weeks after the completion of lengthening. The operated and contralateral tibias were examined with biplanar roentgenography, computerized tomography, and technetium scintigraphy. The torsional strengths of the tibias were then determined and histologic analysis was performed. There was considerable variation in the healing rate of the lengthening sites, both within and among groups. Histologic analysis and results of the imaging modalities revealed different degrees of development of the lengthening sites among members of the same group. Two types of mechanical behavior were seen, corresponding to completely and incompletely ossified lengthening zones. The strongest samples were those that had ossified across their lengthening sites by four or six weeks. The degree of recorticalization of the new bone and the extent of formation of a marrow space correlated well with increased torsional strength. The average strength of the fully ossified bones decreased progressively with time, paralleling a decrease in cortical thickness outside the lengthening zone. This seems to be a consequence of stress shielding by the fixator and disuse by the animal.

Polylactic acid (PLA) homopolymers with molecular weights of 105,000; 21,000; 3300; 650; and 160d (PLA105000, PLA21000, PLA3300, PLA650, and PLA160, respectively) were prepared and evaluated as carriers for bone morphogenetic protein (BMP). Composites consisting of 4 mg of water-soluble, semipurified BMP and 100 mg of one of the PLA homopolymers were implanted into the dorsal muscles of mice. PLA105000/BMP, PLA21000/BMP, PLA3300/BMP, and PLA160/BMP composites failed to induce new bone formation; PLA105000, PLA21000, and PLA3300 elicited foreign-body reactions or chronic inflammation (or both), and PLA160 produced tissue necrosis. PLA650/BMP composites induced cartilage formation within one week and induced bone with hematopoietic marrow at three weeks postimplantation. PLA650/BMP composites were completely absorbed and replaced by new bone. The results suggest that within this group of PLA homopolymers, PLA650 may be the only type suitable for use as a BMP carrier.

Bone development was induced by bone morphogenetic protein (BMP) adsorbed to hydroxyapatite (HAP) under the periosteum of rat parietal bone. The BMP was isolated by Sephacryl-S200 column chromatography after passage through a membrane filter system. The yield of the fraction with BMP activity was 20 times greater than yields obtained by conventional methods. BMP was adsorbed to HAP in vacuo. HAP alone was implanted as a control. The formation of cartilage and bone was observed seven days after the implantation of BMP-HAP, and the newly formed bone fused with the host calvaria after 28 days. In control rats, no bone formation was seen within 56 days after implantation. The BMP-HAP complex was more effective in induced periosteal bone formation than HAP only.

It has been previously shown that osteoinduction by demineralized bone powder (DBP) in the rat decreases as the age of the recipient animal increases. In the present study, the effects of age on osteoinduction by DBP were evaluated in the rat by varying the age of the donor and the recipient animal. Cartilage and bone formation in subcutaneous pouches was assessed using a histologic grading technique in which a composite score was derived from analysis of multiple histologic sections from each specimen. The results confirm the previously reported decrease in osteoinduction in middle-aged adult animals compared with younger ones. However, DBP prepared from middle-aged adult rats was more inductive than that prepared from either prepubertal or young postpubertal animals. The latter result contradicts the widely held belief that demineralized bone matrix from younger animals is more inductive than that from older ones. This finding may help to further elucidate the mechanism of ectopic bone formation and lead to more inductive bone graft substitutes for human use.

To give tenacity to high porosity (80%-90%) hydroxyapatite (HA) and to make a stronger bone substitute, a collagen-coated porous HA (C-HA) was prepared, and its compressive strength was examined. By implanting C-HA into the femoral condyle of adult rabbits, the capacity for new bone formation and foreign-body reactivity were quantitatively compared with those of HA alone. The compressive strength of C-HA was 4.3 times greater than HA. At four weeks after implantation, the mean areal ratio of the newly formed bone in the C-HA block in each rabbit was 9.9%, which was somewhat less than the 13.7% in the HA block; the mean number of multinucleated giant cells (MGC) per visual field at x25 in the C-HA-implanted specimens in each rabbit was 14.4, significantly larger than the 6.1 in the HA specimens. However, 12 weeks after C-HA implantation, the areal ratio of new bone increased to 32.7%, the number of MGC decreased to 9.4, and the differences compared to the values in HA cases, 31.5% and 6.8, disappeared. These results showed that C-HA is mechanically stronger than HA and that there is no difference between HA and C-HA in capacity for new bone formation or foreign-body reaction.

The effect of pulsed electromagnetic stimulation on bone formation was tested in a lower-limb-lengthening model in the rabbit. Limb lengthening was performed by distraction epiphysiolysis. A specially designed external distraction device allowed 10 mm of lengthening of the tibia. Coils to generate a pulsed electromagnetic field were clipped onto the distractor. Stimulation started after a distraction period of three weeks and was continuous for 18 weeks. A control group received the same treatment without stimulation. Bone formation in the elongated zone was evaluated by computed tomography, scintigraphy, and histology. Bone healing involved accretion of callus followed by a process of remodeling, resulting in the formation of a solid cortex. The formation of a diaphysislike structure at the original site of the metaphysis progressed from the distal end of the elongated zone upward. Electromagnetic stimulation had no effect on the rate or extent of bone formation and remodeling.

One hundred forty-six noncemented porous-coated hip and knee implants retrieved from 97 patients were evaluated histologically for the type, amount, and anatomic distribution of tissue ingrowth. The degree of inflammatory cell infiltrate present was also evaluated and the predominant cell type was identified. An inflammatory infiltrate was present in the components of 21 of 97 patients (22%). In 16 of the 21 cases the infiltrate was lymphocytes and histiocytes with a minor population of plasma cells. One of the remaining five cases had a predominately plasma cell reaction, and the other four had significant populations of plasma cells. Vascular proliferation was observed in nine of the 21 cases. Bone ingrowth was present in ten of the 21 cases. A 38% incidence of removal for persistent pain was present in cases with an inflammatory infiltrate. Seventeen of 87 patients (20%) with cobalt-chromium devices and four of ten patients (40%) with titanium devices were identified as having an inflammatory infiltrate. The origin of the inflammatory infiltrate is unclear. All patients with inflammatory infiltrates had noninfected implants, which were not loose roentgenographically or clinically at the time of removal. Hypersensitivity and allergic responses to metal ions may produce such infiltrates. It is impossible, however, in the present study to definitively determine the etiology of the infiltrates.

The effect of onset and duration of growth hormone administration on the biomechanical properties of healing rat tibial fractures was investigated after 40 days of healing. Biosynthetic human growth hormone, 2.7 mg/kg body weight/day, was given in two daily injections to three groups of rats: (1) for the entire healing period; (2) for the first 20 days; and (3) for the last 20 days of healing. Three corresponding groups of control rats were injected with saline. In Group 1, maximum load and stiffness of the healing fractures increased to 165% and 175%, respectively, compared to the control group. In Group 2, maximum load, stiffness, maximum stress, and energy absorption at maximum load increased to 222%, 175%, 171%, and 247%, respectively, compared to the control group. In Group 3, no statistically detectable effects were found. The results show that growth hormone stimulates fracture healing both when given during the first part of the healing period and when given during the entire healing period.