The Dorsal Ligament Complex: A Cadaveric, Histology, and Imaging Study.

PURPOSE: The distinction between the dorsal intercarpal (DIC) and dorsal scaphotriquetral (DST) ligaments is imprecise and unclear in the literature. The purpose of our cadaveric study was to define the origins, insertions, and anatomic relationships of the dorsal wrist ligaments and relate these anatomic findings to magnetic resonance imaging (MRI) scans and histology. METHODS: The study included 17 unmatched fresh-frozen cadaveric specimens (7 male and 10 female), with a mean age of 67.1 years (range, 48-86 years). Wrists with arthritis or carpal malalignment were excluded. Ligaments were dissected and insertion sites were recorded in the radioulnar (width) and proximodistal (length) dimensions, centered at the midpoints of the insertion. Three cadaveric specimens underwent a histologic analysis to demonstrate ligament composition and insertion sites. Three additional cadavers underwent MRI, from which 3-dimensional models were built to model ligament topography. RESULTS: The conjoined triquetral insertion of the DIC, DST, and dorsal radiocarpal (DRC) measured 88.5 ± 6.4 mm2. In each specimen, there were 2 distinct deep and superficial components of intercarpal fibers. The deep component inserted on the lunate with an area of 59.0 ± 5.0 mm2. The deep and superficial components diverged as they coursed radially. The superficial component proceeded to the scaphoid ridge, trapezium, and trapezoid, whereas the deep component inserted on the proximal row. The deep fibers blended distally from their lunate insertion with the DST, forming a robust, 2.9 ± 0.8-mm wide extension over the dorsal capitate. The DRC inserted on the lunate, proximal to the DIC and DST insertions, with an area of 23.9 ± 5.4 mm2. CONCLUSIONS: The dorsal ligament complex forms a firm link across the proximal carpal row and the DST provides extension of the proximal row over the capitate. CLINICAL RELEVANCE: This information can guide surgeons while performing a dorsal approach to the wrist and repairing traumatic ligament disruption.

Carbonyl iron and iron dextran therapies cause adverse effects on bone health in juveniles with chronic kidney disease.

Anemia is a frequent complication of chronic kidney disease (CKD), related in part to the disruption of iron metabolism. Iron therapy is very common in children with CKD and excess iron has been shown to induce bone loss in non-CKD settings, but the impact of iron on bone health in CKD remains poorly understood. Here, we evaluated the effect of oral and parenteral iron therapy on bone transcriptome, bone histology and morphometry in two mouse models of juvenile CKD (adenine-induced and 5/6-nephrectomy). Both modalities of iron therapy effectively improved anemia in the mice with CKD, and lowered bone Fgf23 expression. At the same time, iron therapy suppressed genes implicated in bone formation and resulted in the loss of cortical and trabecular bone in the mice with CKD. Bone resorption was activated in untreated CKD, but iron therapy had no additional effect on this. Furthermore, we assessed the relationship between biomarkers of bone turnover and iron status in a cohort of children with CKD. Children treated with iron had lower levels of circulating biomarkers of bone formation (bone-specific alkaline phosphatase and the amino-terminal propeptide of type 1 procollagen), as well as fewer circulating osteoblast precursors, compared to children not treated with iron. These differences were independent of age, sex, and glomerular filtration rate. Thus, iron therapy adversely affected bone health in juvenile mice with CKD and was associated with low levels of bone formation biomarkers in children with CKD.

A Comparison Between Two Collagen Nerve Conduits and Nerve Autograft: A Rat Model of Motor Nerve Regeneration.

PURPOSE: To compare recovery in a rat model of sciatic nerve injury using a novel polyglycolic acid (PGA) conduit, which contains collagen fibers within the tube, as compared with both a hollow collagen conduit and nerve autograft. We hypothesize that a conduit with a scaffold will provide improved nerve regeneration over hollow conduits and demonstrate no significant differences when compared with autograft. METHODS: A total of 72 Sprague-Dawley rats were randomized into 3 experimental groups, in which a unilateral 10-mm sciatic defect was repaired using either nerve autograft, a hollow collagen conduit, or a PGA collagen-filled conduit. Outcomes were measured at 12 and 16 weeks after surgery, and included bilateral tibialis anterior muscle weight, voltage and force maximal contractility, assessment of ankle contracture, and nerve histology. RESULTS: In all groups, outcomes improved between 12 and 16 weeks. On average, the autograft group outperformed both conduit groups, and the hollow conduit demonstrated improved outcomes when compared with the PGA collagen-filled conduit. Differences in contractile force, however, were significant only at 12 weeks (autograft > hollow collagen conduit > PGA collagen-filled conduit). At 16 weeks, contractile force demonstrated no significant difference but corroborated the same absolute results (autograft > hollow collagen conduit > PGA collagen-filled conduit). CONCLUSIONS: Nerve repair using autograft provided superior motor nerve recovery over the 2 conduits for a 10-mm nerve gap in a murine acute transection injury model. The hollow collagen conduit demonstrated superior results when compared with the PGA collagen-filled conduit. CLINICAL RELEVANCE: The use of a hollow collagen conduit provides superior motor nerve recovery as compared with a PGA collagen-filled conduit.

Heat and radiofrequency plasma glow discharge pretreatment of a titanium alloy: evidence [corrected] for enhanced osteoinductive properties.

It is believed that orthopedic and implant longevity can be improved by optimizing fixation, or direct bone-implant contact, through the stimulation of new bone formation around the implant. The purpose of this study was to determine whether heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of Ti6Al4V stimulated calcium-phosphate mineral formation in cultures of attached MC3T3 osteoprogenitor cells with or without a fibronectin coating. Calcium-phosphate mineral was analyzed by flame atomic absorption spectrophotometry, scanning electron microscopy (SEM)/electron dispersive X-ray microanalysis (EDAX) and Fourier transformed infrared spectroscopy (FTIR). RFGD and heat pretreatments produced a general pattern of increased total soluble calcium levels, although the effect of heat pretreatment was greater than that of RFGD. SEM/EDAX showed the presence of calcium-and phosphorus-containing particles on untreated and treated disks that were more numerous on fibronectin-coated disks. These particles were observed earliest (1 week) on RFGD-pretreated surfaces. FTIR analyses showed that the heat pretreatment produced a general pattern of increased levels of apatite mineral at 2-4 weeks; a greater effect was observed for fibronectin-coated disks compared to uncoated disks. The observed findings suggest that heat pretreatment of Ti6Al4V increased the total mass of the mineral formed in MC3T3 osteoprogenitor cell cultures more than RFGD while the latter pretreatment hastened the early deposition of mineral. These findings help to support the hypothesis that the pretreatments enhance the osteoinductive properties of the alloy.

Conditional inactivation of the CXCR4 receptor in osteoprecursors reduces postnatal bone formation due to impaired osteoblast development.

Cysteine (C)-X-C motif chemokine receptor 4 (CXCR4), the primary receptor for stromal cell-derived factor-1 (SDF-1), is involved in bone morphogenic protein 2 (BMP2)-induced osteogenic differentiation of mesenchymal progenitors. To target the in vivo function of CXCR4 in bone and explore the underlying mechanisms, we conditionally inactivated CXCR4 in osteoprecursors by crossing osterix (Osx)-Cre mice with floxed CXCR4 (CXCR4(fl/fl)) mice to generate knock-outs with CXCR4 deletion driven by the Osx promoter (Osx::CXCR4(fl/fl)). The Cre-mediated excision of CXCR4 occurred exclusively in bone of Osx::CXCR4(fl/fl) mice. When compared with littermate controls, Osx::CXCR4(fl/fl) mice developed smaller osteopenic skeletons as evidenced by reduced trabecular and cortical bone mass, lower bone mineral density, and a slower mineral apposition rate. In addition, Osx::CXCR4(fl/fl) mice displayed chondrocyte disorganization in the epiphyseal growth plate associated with decreased proliferation and collagen matrix syntheses. Moreover, mature osteoblast-related expression of type I collagen α1 and osteocalcin was reduced in bone of Osx::CXCR4(fl/fl) mice versus controls, suggesting that CXCR4 deficiency results in arrested osteoblast progression. Primary cultures for osteoblastic cells derived from Osx::CXCR4(fl/fl) mice also showed decreased proliferation and impaired osteoblast differentiation in response to BMP2 or BMP6 stimulation, and suppressed activation of intracellular BMP receptor-regulated Smads (R-Smads) and Erk1/2 was identified in CXCR4-deficient cells and bone tissues. These findings provide the first in vivo evidence that CXCR4 functions in postnatal bone development by regulating osteoblast development in cooperation with BMP signaling. Thus, CXCR4 acts as an endogenous signaling component necessary for bone formation.

Bone loss caused by iron overload in a murine model: importance of oxidative stress.

Osteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Improved specificity of cartilage matrix evaluation using multiexponential transverse relaxation analysis applied to pathomimetically degraded cartilage.

The noninvasive early detection of specific matrix alterations in degenerative cartilage disease would be of substantial use in basic science studies and clinically, but remains an elusive goal. Recently developed MRI methods exhibit some specificity, but require contrast agents or nonstandard pulse sequences and hardware. We present a multiexponential approach which does not require contrast agents or specialized hardware, and uses a standard multiple-echo spin-echo sequence. Experiments were performed on tissue models of degenerative cartilage using enzymes with distinct actions. MR results were validated using histologic, biochemical and infrared spectroscopic analyses. The sulfated glycosaminoglycan per dry weight (dw) in bovine nasal cartilage was 0.72 ± 0.06 mg/mg dw and was reduced through chondroitinase AC and collagenase digestion to 0.56 ± 0.12 and 0.58 ± 0.13 mg/mg dw, respectively. Multiexponential analysis of data obtained at 9.4 T permitted the identification of tissue compartments assigned to the proteoglycan component of the matrix and to bulk water. Enzymatic treatment resulted in a significant reduction in the ratio of proteoglycan-bound to free water from 0.13 ± 0.02 in control cartilage to 0.03 ± 0.02 and 0.05 ± 0.06 under chondroitinase AC and collagenase treatment, respectively. As expected, monoexponential T(2) increased with both degradation protocols, but without further specificity to the nature of the degradation. An important eventual extension of this approach may be to map articular cartilage degeneration in the clinical setting. As an initial step towards this, localized multiexponential T(2) analysis was performed on control and trypsin treated excised bovine patella. The results obtained on this articular cartilage sample were readily interpretable in terms of proteoglycan-associated and relatively free water compartments. In potential clinical applications, signal-to-noise ratio constraints will define the threshold for the detection of macromolecular compartment changes at a given spatial scale. The multiexponential approach has potential application to the early detection of cartilage degradation with the use of appropriate pulse parameters under high signal-to-noise ratio conditions.

Characterization of the structure-function relationship at the ligament-to-bone interface.

Soft tissues such as ligaments and tendons integrate with bone through a fibrocartilaginous interface divided into noncalcified and calcified regions. This junction between distinct tissue types is frequently injured and not reestablished after surgical repair. Its regeneration is also limited by a lack of understanding of the structure-function relationship inherent at this complex interface. Therefore, focusing on the insertion site between the anterior cruciate ligament (ACL) and bone, the objectives of this study are: (i) to determine interface compressive mechanical properties, (ii) to characterize interface mineral presence and distribution, and (iii) to evaluate insertion site-dependent changes in mechanical properties and matrix mineral content. Interface mechanical properties were determined by coupling microcompression with optimized digital image correlation analysis, whereas mineral presence and distribution were characterized by energy dispersive x-ray analysis and backscattered scanning electron microscopy. Both region- and insertion-dependent changes in mechanical properties were found, with the calcified interface region exhibiting significantly greater compressive mechanical properties than the noncalcified region. Mineral presence was only detectable within the calcified interface and bone regions, and its distribution corresponds to region-dependent mechanical inhomogeneity. Additionally, the compressive mechanical properties of the tibial insertion were greater than those of the femoral. The interface structure-function relationship elucidated in this study provides critical insight for interface regeneration and the formation of complex tissue systems.

Differentiation and mineralization of murine mesenchymal C3H10T1/2 cells in micromass culture.

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. (45)Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of (45)Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.

Evaluation of a porous polyurethane scaffold in a partial meniscal defect ovine model.

PURPOSE: The objective of this study was to assess the performance of a degradable porous polyurethane scaffold in a partial meniscectomy ovine model. METHODS: We subjected 42 skeletally mature ewes to unilateral partial excision of the lateral meniscus. In 19 animals the defect was left unfilled; in 23 animals a scaffold was inserted. Knees were examined by magnetic resonance imaging, gross inspection, and histologic inspection of the cartilage of the tibial plateau. RESULTS: In contrast to what has been previously reported in a complete meniscal replacement model, cartilage damage did not occur under the site of scaffold implantation; this was likely influenced by the rapid infiltration of cells and the dense tissue that formed within the scaffold. Cartilage damage in both groups was located close to the midline of the joint. No significant difference in the condition of the articular cartilage of the tibial plateau was seen between groups up to 12 months postoperatively. This result was influenced by the fact that the partly meniscectomized knees also showed unexpected tissue regeneration within the defect site, which raises concern about the suitability of using a partial meniscectomy as a control in the ovine model. CONCLUSIONS: Our study has shown that implantation of a polyurethane scaffold in a partial meniscectomy ovine model promotes tissue ingrowth without damaging the cartilage with which it articulates. CLINICAL RELEVANCE: Meniscal deficiency is a common occurrence, the effective clinical management of which is limited by the absence of an off-the-shelf implantable construct.

Ablation of cathepsin k activity in the young mouse causes hypermineralization of long bone and growth plates.

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.

Characterization of dystrophic calcification induced in mice by cardiotoxin.

Dystrophic calcifications often occur after injury, infection, or onset of certain rheumatic diseases. Treatment has been limited to surgical removal following failure of medical therapy. In an attempt to establish a reproducible animal model for dystrophic calcification that permitted the screening of potential interventions, we evaluated cardiotoxin (injury)-induced calcifications in three murine strains at both the cellular and ultrastructural levels. All osteopontin null mice and tumor necrosis factor receptor null mice on a C57B6 background had calcifications at days 3 and 7 after injury compared to 75% of wild-type C57B6 mice. There was no difference in mineral content among calcifications from the three mouse strains. Osteogenesis was suggested by the expression of osteocalcin, osterix, and alkaline phosphatase in calcified murine muscle tissue. Osteoclast-like cells facilitated the removal of transient dystrophic deposits (<28 days) in all models. However, none of the models showed an association of mineral crystals with collagen, suggesting that the deposits were not bone-like. The dystrophic mechanism was validated as cell death, and mitochondrial calcifications occurred soon after skeletal muscle injury in the three murine strains.

Botox-induced muscle paralysis alters intracortical porosity and osteocyte lacunar density in skeletally mature rats.

Reduced mechanical loading can lead to disuse osteoporosis, resulting in bone fragility. Disuse models report macroscopic bone loss due to muscle inactivity and immobilization, yet only recently has there been quantification of the effects of disuse on the vascular pores and osteocyte network, which are believed to play an important role in mechanotransduction via interstitial fluid flow. The goal of this study was to perform a high-resolution analysis of the effects of muscle inactivity on intracortical porosity and osteocyte lacunar density in skeletally mature rats. Muscle paralysis was induced in 20-week-old female Sprague Dawley rats by injection of botulinum neurotoxin. Rats were injected in the right hindlimb muscles with either Botox (BTX, n = 8) or saline solution (CTRL, n = 8), with a third group used as baseline controls (n = 8). Four weeks after injection, Botox caused a ∼60% reduction in hindlimb muscle mass. High-resolution micro-CT analysis showed that Botox-induced muscle paralysis increased vascular canal porosity and reduced osteocyte lacunar density within the tibial metaphysis cortex. Cortical thickness and other areal properties were diminished in the proximal tibial metaphysis, whereas no differences were found in the mid-diaphysis. Within the BTX group, the injected limbs showed a lower cancellous bone volume fraction relative to the contralateral limb. These results indicate that diminished muscle activity alters the vascular canal porosity and osteocyte lacunar density in cortical bone, which could alter interstitial fluid flow, affecting molecular transport and the transmission of mechanical signals to osteocytes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Polymeric mesh and insulin-like growth factor 1 delivery enhance cell homing and graft-cartilage integration.

Cartilage injury, such as full-thickness lesions, predisposes patients to the premature development of osteoarthritis, a degenerative joint disease. While surgical management of cartilage lesions has improved, long-term clinical efficacy has stagnated, owing to the lack of hyaline cartilage regeneration and inadequate graft-host integration. This study tests the hypothesis that integration of cartilage grafts with native cartilage can be improved by enhancing the migration of chondrocytes across the graft-host interface via the release of chemotactic factor from a degradable polymeric mesh. To this end, a polylactide-co-glycolide/poly-ε-caprolactone mesh was designed to localize the delivery of insulin-like growth factor 1 (IGF-1), a well-established chondrocyte attractant. The release of IGF-1 (100 ng/mg) enhanced cell migration from cartilage explants, and the mesh served as critical structural support for cell adhesion, growth, and production of a cartilaginous matrix in vitro, which resulted in increased integration strength compared with mesh-free repair. Further, this neocartilage matrix was structurally contiguous with native and grafted cartilage when tested in an osteochondral explant model in vivo. These results demonstrate that this combined approach of a cell homing factor and supportive matrix will promote cell-mediated integrative cartilage repair and improve clinical outcomes of cartilage grafts in the treatment of osteoarthritis.

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures.

Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb-bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4 mM inorganic phosphate and no organic-phosphate esters; control cultures had 1 mM inorganic phosphate. Mineralization was monitored based on (45)Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis. Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin-LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating that the phosphatases were, in part, providing a source of inorganic phosphate. To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.

Generalized metabolic bone disease in Neurofibromatosis type I.

Skeletal abnormalities are a recognized component of Neurofibromatosis type I (NF1) but a generalized metabolic bone defect in NF1 has not been fully characterized thus far. The purpose of this study was to characterize at the densitometric, biochemical and pathological level the bone involvement in NF1 patients. Using dual energy X-ray absorptiometry (DXA) we analyzed bone status in 73 unselected NF1 subjects, 26 males and 47 females, mainly children and adolescents (mean age: 16.6 years). In a subgroup of subjects with low bone mass, we measured indices of calcium-phosphate metabolism, bone turnover, and bone density before and after vitamin D and calcium treatment. We found statistically significant and generalized reduction in bone mass with the mean lumbar bone mineral density (BMD) z-score being -1.38+/-1.05 (CI 95% -1.62 to -1.13), and whole body bone mineral content (BMC) z-score -0.61+/-1.19 (CI 95% -0.94 to -0.29), both significantly reduced compared to normal controls (p<.001). PTH was moderately elevated and after 4 months of supplemental therapy with calcium and vitamin D, it decreased to the normal range. However, BMD z-scores did not significantly improve after 2 years of follow-up. Histological analysis of bone samples from NF1 patients revealed substantial alteration of bone microarchitecture due mainly to reduced trabecular bone. Our observations are consistent with a generalized bone metabolic defect due to loss of the function of neurofibromin. Early identification of patients with osteoporosis may permit more timely and aggressive treatments to prevent the likely substantial morbidity associated with increased fracture risk later in life.

Integrating soft and hard tissues via interface tissue engineering.

The enthesis, or interface between bone and soft tissues such as ligament and tendon, is prone to injury and often does not heal, even post surgical intervention. Interface tissue engineering represents an integrative strategy for regenerating the native enthesis by functionally connecting soft and hard tissues and thereby improving clinical outcome. This review focuses on integrative and cell-instructive scaffold designs that target the healing of the two most commonly injured soft tissue-bone junctions: tendon-bone interface (e.g., rotator cuff) and ligament-bone interface (e.g., anterior cruciate ligament). The inherent connectivity between soft and hard tissues is instrumental for musculoskeletal motion and is therefore a key design criterion for soft tissue regeneration. To this end, scaffold design for soft tissue regeneration have progressed from single tissue systems to the emerging focus on pre-integrated and functional composite tissue units. Specifically, a multifaceted, bioinspired approach has been pursued wherein scaffolds are tailored to stimulate relevant cell responses using spatially patterned structural and chemical cues, growth factors, and/or mechanical stimulation. Moreover, current efforts to elucidate the essential scaffold design criteria via strategic biomimicry are emphasized as these will reduce complexity in composite tissue regeneration and ease the related burden for clinical translation. These innovative studies underscore the clinical relevance of engineering connective tissue integration and have broader impact in the formation of complex tissues and total joint regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1069-1077, 2018.

The effects of estrogen deficiency on cortical bone microporosity and mineralization.

Recent studies have demonstrated matrix-mineral alterations in bone tissue surrounding osteocytes in estrogen-deficient animals. While cortical bone porosity has been shown to be a contributor to the mechanical properties of bone tissue, little analysis has been done to investigate the effects of estrogen deficiency on bone's microporosities, including the vascular and osteocyte lacunar porosities. In this study we examined alterations in cortical bone microporosity, mineralization, and cancellous bone architecture due to estrogen deficiency in the ovariectomized rat model of postmenopausal osteoporosis. Twenty-week-old female Sprague-Dawley rats were subjected to either ovariectomy or sham surgery. Six weeks post-surgery tibiae were analyzed using high-resolution micro-CT, backscattered electron imaging, nanoindentation, and dynamic histomorphometry. Estrogen deficiency caused an increase in cortical bone vascular porosity, with enlarged vascular pores and little change in tissue mineral density in the proximal tibial metaphysis. Measurements of cancellous architecture corresponded to previous studies reporting a decrease in bone volume fraction, an increase in trabecular separation, and a decrease in trabecular number in the proximal tibia due to estrogen deficiency. Nanoindentation results showed no differences in matrix stiffness in osteocyte-rich areas of the proximal tibia of estrogen-deficient rats, and bone labeling and backscattered electron imaging showed no significant changes in mineralization around the vascular pores. The findings demonstrate local surface alterations of vascular pores due to estrogen deficiency. An increase in cortical vascular porosity may diminish bone strength as well as alter bone mechanotransduction via interstitial fluid flow, both of which could contribute to bone fragility during postmenopausal osteoporosis.

Compositional mapping of the mature anterior cruciate ligament-to-bone insertion.

The anterior cruciate ligament (ACL)-to-bone interface constitutes a complex, multi-tissue structure comprised of contiguous ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL-to-bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non-mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513-2523, 2017.

Zoledronic acid improves bone histomorphometry in a murine model of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder predominately affecting young females, caused by deficiency of the global transcriptional protein methyl CpG binding protein 2 (MeCP2). Osteoblasts express MeCP2 and girls with RTT experience early onset osteoporosis, decreased bone mass and an increased fracture risk. There is no defined treatment for osteoporosis associated with RTT. The present study evaluated the effects of zoledronic acid (ZA), a third generation nitrogen-containing bisphosphonate with primarily anti-osteoclastic activity, in a mouse model of MeCP2 deficiency. Mice received weekly injections of 20µg/kg ZA for six weeks. Due to the shortened lifespan of hemizygous male (Mecp2-null) mice, treatment began at 3weeks of age for this group and corresponding wildtype (WT) male mice. Treatment for heterozygous (HET) and WT female mice began at 8weeks of age. Micro-computed tomography (micro-CT) and dynamic analyses of bone turnover were performed. ZA treatment led to significant increases in bone volume fraction, number, connectivity density and apparent density of trabecular bone in all genotypes of mice. In contrast, cortical bone generally was unaffected by ZA injections. Parameters of bone turnover, including mineral apposition rate, labeled bone surface and bone formation rate decreased after treatment with ZA. Mecp2-null mice had reduced labeled bone surface and bone formation rate compared to WT male mice. The results indicate that ZA treatment significantly improved trabecular bone mass in a murine model of RTT with little effect on cortical bone.