Subperiosteal delivery of transforming growth factor beta 1 and human growth hormone from mineralized PCL films.

The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provide a novel means by which to improve fracture healing, treat neoplasms or infections, or modulate growth. In this study, we constructed single-sided mineral-coated poly-ε-caprolactone membranes capable of binding and releasing transforming growth factor beta 1 (TGF-ß1) and human growth hormone (hGH). After demonstrating biological activity in vitro and characterization of their release, these thin bioabsorbable membranes were surgically implanted using an immature rabbit model. Membranes were circumferentially wrapped under the periosteum, thus placed in direct contact with the proximal metaphysis to assess its bioactivity in vivo. The direct effects on the metaphyseal bone, bone marrow, and overlying periosteum were assessed using radiography and histology. Effects of membrane placement at the tibial growth plate were assessed via physeal heights, tibial growth rates (pulsed fluorochrome labeling), and tibial lengths. Subperiosteal placement of the mineralized membranes induced greater local chondrogenesis in the plain mineral and TGF-ß1 samples than the hGH. More exuberant and circumferential ossification was seen in the TGF-ß1 treated tibiae. The TGF-ß1 membranes also induced hypocellularity of the bone marrow with characteristics of gelatinous degeneration not seen in the other groups. While the proximal tibial growth plates were taller in the hGH treated than TGF-ß1, no differences in growth rates or overall tibial lengths were found. In conclusion, these data demonstrate the feasibility of using bioabsorbable mineral coated membranes to deliver biologically active compounds subperiosteally in a sustained fashion to affect cells at the insertion site, bone marrow, and even growth plate.

Microwave Ablation of the Pig Growth Plate: Proof of Concept for Minimally Invasive Epiphysiodesis.

BACKGROUND: Different surgical methods for epiphysiodesis of limb length discrepancy (LLD) have been described. Although these methods are variably effective, they are associated with morbidity (pain and limp) and potential complications. Microwave ablation is a less-invasive opportunity to halt growth by selectively destroying the growth plate via thermal energy to treat LLD in children. QUESTIONS/PURPOSES: In this proof-of-concept study using an in vivo pig model, we asked: (1) What is the durability of response 2 to 4 months after microwave ablation of the tibial growth plate as measured by length and angulation of the tibia via a CT scan? (2) Was articular cartilage maintained as measured by standard histologic staining for articular cartilage viability? METHODS: To develop an in vivo protocol for microwave ablation, we placed microwave antennas adjacent to the proximal tibia growth plate in the cadaveric hindlimbs of 18 3-month-old pigs. To determine the suitable time, we varied ablation from 90 to 270 seconds at 65-W power settings. After sectioning the tibia, we visually assessed for discoloration (implying growth plate destruction) that included the central growth plate but did not encroach into the epiphysis in a manner that could disrupt the articular surface. Using this information, we then performed microwave ablation on three live female pigs (3.5 to 4 months old) to evaluate physiologic changes and durability of response. A postprocedure MRI was performed to ensure the intervention led to spatial growth plate alterations similar to that seen in cadavers. This was followed by serial CT, which was used to assess the potential effect on local bone and growth until the animals were euthanized 2 to 4 months after the procedure. We analyzed LLD, angular deformity, and bony deformity using CT scans of both tibias. The visibility of articular cartilage was compared with that of the contralateral tibia via standard histologic staining, and growth rates of the proximal tibial growth plate were compared via fluorochrome labeling. RESULTS: Eighteen cadaveric specimens showed ablation zones across the growth plate without visual damage to the articular surface. The three live pigs did not exhibit changes in gait or require notable pain medication after the procedure. Each animal demonstrated growth plate destruction, expected limb shortening (0.8, 1.2, and 1.5 cm), and bony cavitation around the growth plate. Slight valgus bone angulation (4º, 5º, and 12º) compared with the control tibia was noted. No qualitatively observable articular cartilage damage was encountered from the histologic comparison with the contralateral tibia for articular cartilage thickness and cellular morphology. CONCLUSION: A microwave antenna placed into a pig's proximal tibia growth plate can slow the growth of the tibia without apparent pain and alteration of gait and function. CLINICAL RELEVANCE: Further investigation and refinement of our animal model is ongoing and includes shorter ablation times and comparison of dynamic ablation (moving the antennae during the ablation) as well as static ablation of the tibia from a medial and lateral portal. These refinements and planned comparison with standard mechanical growth arrest in our pig model may lead to a similar approach to ablate growth plates in children with LLD.

Periostin modulates extracellular matrix behavior in tendons.

Periostin, originally named osteoblast-specific factor 2 (OSF-2) has been identified primarily in collagen rich, biomechanically active tissues where its role has been implicated in mechanisms to maintain the extracellular matrix (ECM), including collagen fibrillogenesis and crosslinking. It is well documented that periostin plays a role in wound healing and scar formation after injury, in part, by promoting cell proliferation, myofibroblast differentiation, and/or collagen fibrillogenesis. Given the significance of periostin in other scar forming models, we hypothesized that periostin will influence Achilles tendon healing by modulating ECM production. Therefore, the objective of this study was to elucidate the effects of periostin during Achilles tendon healing using periostin homozygous (Postn -/-) and heterozygous (Postn +/-) mouse models. A second experiment was included to further examine the influence of periostin on collagen composition and function using intact dorsal tail tendons. Overall, Postn -/- and Postn +/- Achilles tendons exhibited impaired healing as demonstrated by delayed wound closure, increased type III collagen production, decreased cell proliferation, and reduced tensile strength. Periostin ablation also reduced tensile strength and stiffness, and altered collagen fibril distribution in the intact dorsal tail tendons. Achilles tendon outcomes support our hypothesis that periostin influences healing, while tail tendon results indicate that periostin also affects ECM morphology and behavior in mouse tendons.

Epigenetic regulation of BAF60A determines efficiency of miniature swine iPSC generation.

Miniature pigs are an ideal animal model for translational research to evaluate stem cell therapies and regenerative applications. While the derivation of induced pluripotent stem cells (iPSCs) from miniature pigs has been demonstrated, there is still a lack of a reliable method to generate and maintain miniature pig iPSCs. In this study, we derived iPSCs from fibroblasts of Wisconsin miniature swine (WMS), Yucatan miniature swine (YMS), and Göttingen minipigs (GM) using our culture medium. By comparing cells of the different pig breeds, we found that YMS fibroblasts were more efficiently reprogrammed into iPSCs, forming colonies with well-defined borders, than WMS and GM fibroblasts. We also demonstrated that YMS iPSC lines with a normal pig karyotype gave rise to cells of the three germ layers in vitro and in vivo. Mesenchymal stromal cells expressing phenotypic characteristics were derived from established iPSC lines as an example of potential applications. In addition, we found that the expression level of the switch/sucrose nonfermentable component BAF60A regulated by STAT3 signaling determined the efficiency of pig iPSC generation. The findings of this study provide insight into the underlying mechanism controlling the reprogramming efficiency of miniature pig cells to develop a viable strategy to enhance the generation of iPSCs for biomedical research.

Histological and radiographic evaluation of three common tendon transfer techniques in an un-ossified bone porcine model: implications for early anterior tibialis tendon transfers in children with clubfeet.

PURPOSE: To compare the histological healing and radiographic effects of tendons transferred to ossified or unossified bone using different tendon fixation techniques. METHODS: Nine new-born piglets underwent bilateral tendon transfers to either the ossified boney calcaneal body or unossified apophysis. The tendons were fixed using metallic suture anchors, sutures alone or a bone tunnel. At six weeks of age, calcanei were harvested, radiologically imaged and then prepared for histology. A semi-quantitative aggregated scoring system with values ranging from 0 (poor) to 15 (excellent), was used to grade healing at the surgical enthesis and the apophyseal ossification was graded by five independent reviewers in triplicate using a modified (1 to 4) validated scoring system. RESULTS: Histologically, the cartilaginous transfers utilizing the tunnel and suture techniques also demonstrated the best average aggregated scores of entheses healing rivalling that measured in transfers using the classic bone tunnel technique (clinical benchmark), whereas suture anchor fixation demonstrated the worst healing in both the ossified and unossified samples. All three transfer techniques caused at least minor alterations in apophyseal ossification, with the most significant changes observed in the metallic suture anchor cohort. The tunnel and suture techniques demonstrated similar and more mild abnormalities in ossification. CONCLUSION: Tendon transfers to unossified bone heal histologically as well as transfers classically performed through tunnels in bone. Suture fixation or tunnel techniques appear radiographically and histologically superior to suture anchors in our newborn porcine model.

Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration.

Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.

Tendon-to-Bone Healing in a Rat Extra-articular Bone Tunnel Model: A Comparison of Fresh Autologous Bone Marrow and Bone Marrow-Derived Mesenchymal Stem Cells.

BACKGROUND: Despite widespread acceptance of fresh autologous bone marrow (BM) for use in clinical practice, limited information exists to analyze if tendon-to-bone healing could be accelerated with local use of fresh autologous BM. PURPOSE: To investigate the effect of fresh autologous BM on tendon-to-bone healing with a novel rat model. STUDY DESIGN: Controlled laboratory study. METHODS: An extra-articular bone tunnel was created and filled with an autologous tendon graft in skeletally mature Sprague-Dawley rats (N = 60). They were then randomly divided into 3 groups: BM group (injection of fresh autologous BM into the tendon-bone interface, n = 20), BM-derived mesenchymal stem cell (BMSC) group (injection of allogenic cultured BMSCs, n = 20), and the control group (tendon-bone interface without injection of BM or BMSCs, n = 20). Biomechanical, histological, and immunohistochemical analyses were performed at 2 and 6 weeks after surgery. RESULTS: The BM group showed a relatively well-organized and dense connective tissue interface with better orientation of collagen fibers as compared with the BMSC group. At 2 weeks, the tendon-bone interface tissue thickness of the BMSC group was 140 ± 25 µm (mean ± SEM), which was significantly greater than the BM group (58 ± 15 µm). The BM group showed fewer M1 macrophages at the tendon-bone interface at 2 and 6 weeks (P < .001). In contrast, there were more M2 macrophages at the interface in the BM group 2 and 6 weeks postoperatively when compared with controls and the BMSC group (P < .001). Biomechanical tests revealed significantly higher stiffness in the BM group versus the control and BMSC groups at 2 and 6 weeks after surgery (P < .05). Load to failure showed similar trends to stiffness. CONCLUSION: These findings indicate that local delivery of fresh autologous BM enhances tendon-to-bone healing better than the alternative treatments in this study. This effect may be partially due to the observed modulation of inflammatory processes, especially in M2 macrophage polarization. CLINICAL RELEVANCE: Fresh autologous BM could be a treatment option for this disorder.

Bone Morphogenetic Protein-6 Attenuates Type 1 Diabetes Mellitus-Associated Bone Loss.

Patients with type 1 diabetes mellitus (T1DM) often suffer from osteopenia or osteoporosis. Although most agree that T1DM-induced hyperglycemia is a risk factor for progressive bone loss, the mechanisms for the link between T1DM and bone loss still remain elusive. In this study, we found that bone marrow-derived mesenchymal stem cells (BMSCs) isolated from T1DM donors were less inducible for osteogenesis than those from non-T1DM donors and further identified a mechanism involving bone morphogenetic protein-6 (BMP6) that was produced significantly less in BMSCs derived from T1DM donors than that in control cells. With addition of exogenous BMP6 in culture, osteogenesis of BMSCs from T1DM donors was restored whereas the treatment of BMP6 seemed not to affect non-T1DM control cells. We also demonstrated that bone mineral density (BMD) was reduced in streptozotocin-induced diabetic mice compared with that in control animals, and intraperitoneal injection of BMP6 mitigated bone loss and increased BMD in diabetic mice. Our results suggest that bone formation in T1DM patients is impaired by reduction of endogenous BMP6, and supplementation of BMP6 enhances osteogenesis of BMSCs to restore BMD in a mouse model of T1DM, which provides insight into the development of clinical treatments for T1DM-assocaited bone loss. Stem Cells Translational Medicine 2019;8:522-534.

Maternal Diets Deficient in Vitamin D Increase the Risk of Kyphosis in Offspring: A Novel Kyphotic Porcine Model.

BACKGROUND: The purpose of this study was to explore the role of perinatal vitamin-D intake on the development and characterization of hyperkyphosis in a porcine model. METHODS: The spines of 16 pigs were assessed at 9, 13, and 17 weeks of age with radiography and at 17 weeks with computed tomography (CT), magnetic resonance imaging (MRI), histology, and bone-density testing. An additional 169 pigs exposed to 1 of 3 maternal dietary vitamin-D levels from conception through the entire lactation period were fed 1 of 4 nursery diets supplying different levels of vitamin D, calcium, and phosphorus. When the animals were 13 weeks of age, upright lateral spinal radiography was performed with use of a custom porcine lift and sagittal Cobb angles were measured in triplicate to determine the degree of kyphosis in each pig. RESULTS: The experimental animals had significantly greater kyphotic sagittal Cobb angles at all time points when compared with the control animals. These hyperkyphotic deformities demonstrated no significant differences in Hounsfield units, contained a slightly lower ash content (46.7% ± 1.1% compared with 50.9% ± 1.6%; p < 0.001), and demonstrated more physeal irregularities. Linear mixed model analysis of the measured kyphosis demonstrated that maternal diet had a greater effect on sagittal Cobb angle than did nursery diet and that postnatal supplementation did not completely eliminate the risk of hyperkyphosis. CONCLUSIONS: Maternal diets deficient in vitamin D increased the development of hyperkyphosis in offspring in this model. CLINICAL RELEVANCE: This study demonstrates that decreased maternal dietary vitamin-D intake during pregnancy increases the risk of spinal deformity in offspring. In addition, these data show the feasibility of generating a large-animal spinal-deformity model through dietary manipulation alone.

Impacts of Interleukin-17 Neutralization on the Inflammatory Response in a Healing Ligament.

In this study, we sought to improve ligament healing by modulating the inflammatory response after acute injury through the neutralization of Interleukin-17 (IL-17), which we hypothesized would decrease inflammatory cell infiltration and cytokine production. Administration of an Interleukin-17 neutralizing antibody (IL-17 NA) immediately following a rat medial collateral ligament (MCL) transection resulted in alterations in inflammatory cell populations and cytokine expression within the healing ligament, but did not reduce inflammation. Specifically, treatment resulted in a decrease in M2 (anti-inflammatory) macrophages, an increase in T cells, and an increase in the levels of IL-2, IL-6, and IL-12 in the MCL 7 days post injury. IL-17NA treatment, and subsequent immunomodulation, did not result in improved ligament healing, as measured by collagen composition and wound size.

Level-specific amputations and resulting regenerative outcomes in the mouse distal phalanx.

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

Tendon transfer to unossified bone in a porcine model: potential implications for early tibialis anterior tendon transfers in children with clubfeet.

PURPOSE: Tibialis anterior tendon transfers (TATT) are commonly performed in young children following Ponseti casting for clubfeet. The classic TATT involves advancing the tendon through a hole drilled in the ossified cuneiform. The aim of this study was to determine if tendons transferred through unossified bones have untoward effects on subsequent bone development. METHOD: Twenty-five piglets underwent one of five surgical procedures. An 18-gauge needle was then used to place a tunnel through the bony or cartilaginous portion of the calcaneus (through direct visualization) and isolated slips of the flexor digitorum superficialis (FDS) were placed through the tunnels, as determined by surgical procedure. Radiographic and/or histologic evaluations of the calcaneal apophyses were then performed. A discrete (1-4) and dichotomous "Normal" or "Abnormal" scoring system was developed and its reliability assessed to grade the appearance of the calcanei. Calcaneal appearances following the surgical procedures were then compared with controls. The average load to failure of a subset of transferred tendons was then compared using an MTS machine. RESULTS: The proposed apophyseal grading system (1-4) demonstrated an intraclass correlational coefficient (ICC) for consistency of 0.92 [95% confidence interval (CI) 0.88 < ICC < 0.95] and ICC for agreement of 0.91 (95% CI 0.86 < ICC < 0.95), indicating strong agreement and consistency. Similarly, Fleiss' kappa for the 1-4 scoring system was found to be 0.67, indicating substantial agreement between reviewers. When the 1-4 system was translated into the dichotomous scheme "Normal" and "Abnormal", the kappa value increased to 0.94, indicating strong agreement. Forty-six apophyses (13 control and 33 operative) were assessed using this scoring scheme. Apophyseal transfers were significantly more abnormal than controls (p < 0.0001), while no difference in abnormalities was found following tunnel placement alone (p = 1). Mechanical testing of the tendons transferred to bone or through the cartilaginous apophysis demonstrated no significant differences (p = 0.2). CONCLUSION: Tendon transfers through unossified bones altered subsequent bone development. SIGNIFICANCE: While the long-term consequence of these structural changes is unknown, these findings suggest that tendon transfers through unossified bones should be avoided and alternative methods of tendon fixation explored.

Growth Retardation (Hemiepiphyseal Stapling) and Growth Acceleration (Periosteal Resection) as a Method to Improve Guided Growth in a Lamb Model.

BACKGROUND: Guided growth corrects pediatric limb deformity by inhibiting growth on the convexity of the bone. Both modular and rigid implants have been used; we endeavor to determine whether a clear advantage of one implant exists. We further hypothesize that improved correction could be realized by accelerating growth with resection of the periosteum. METHODS: Sixteen lambs underwent guided growth of the medial proximal tibia (the opposite limb served as a control). Group 1 used a rigid staple (n=5); group 2 a modular plate and screw construct (n=5), and group 3 had a similar device plus periosteal resection (n=6). Radiographs tracked the progression of deformity in the coronal plane. Before sacrifice, pulsed fluorochrome labels allowed for temporal and spatial growth rate analysis. At sacrifice, True Deformity was calculated (and compared with control tibia) from standardized radiographs in the coronal and sagittal planes. Device Efficiencies were normalized by dividing True Deformity produced (degrees) by the Expected Growth gain (mm) from the control limb. RESULTS: Group 3 produced greater coronal plane deformity than group 1 by an average of 2.2 degrees per month (P=0.001) and group 2 by an average of 2.4 degrees per month (P=0.0007). At sacrifice, groups 1 and 2 were equally effective at limiting growth to 75% of control; no differences in growth retardation were noted. No differences in Device Efficiency were noted between groups 1 and 2. The Device Efficiency was significantly different between groups 1 and 2 with comparison with group 3 (P=0.05 and P=0.022); with a 2.5 degree/mm faster deformation in the stripped cohort. CONCLUSIONS: Rigid implants initially produced deformity quicker than modular constructs; yet ultimately, both implants were equally effective at guiding growth. Device Efficiency for the modular group improved significantly with the addition of periosteal stripping as method to accelerate growth.

Primed Mesenchymal Stem Cells Alter and Improve Rat Medial Collateral Ligament Healing.

Cell therapy with mesenchymal stem cells (MSCs) can improve tissue healing. It is possible, however, that priming MSCs prior to implantation can further enhance their therapeutic benefit. This study was then performed to test whether priming MSCs to be more anti-inflammatory would enhance healing in a rat ligament model, i.e. a medial collateral ligament (MCL). MSCs were primed for 48 h using polyinosinic acid and polycytidylic acid (Poly (I:C)) at a concentration of 1 µg/ml. Rat MCLs were surgically transected and administered 1 × 10(6) cells in a carrier solution at the time of injury. A series of healing metrics were analyzed at days 4 and 14 post-injury in the ligaments that received primed MSCs, unprimed MSCs, or no cells (controls). Applying primed MSCs beneficially altered healing by affecting endothelialization, type 2 macrophage presence, apoptosis, procollagen 1α, and IL-1Ra levels. When analyzing MSC localization, both primed and unprimed MSCs co-localized with endothelial cells and pericytes suggesting a supportive role in angiogenesis. Priming MSCs prior to implantation altered key ligament healing events, resulted in a more anti-inflammatory environment, and improved healing.

Periosteal Fiber Transection During Periosteal Procedures Is Crucial to Accelerate Growth in the Rabbit Model.

BACKGROUND: Disruption of the periosteum has been used to explain overgrowth after long bone fractures. Clinically, various periosteal procedures have been reported to accelerate growth with varied results. Differences between procedures and study populations, in these prior studies, make drawing conclusions regarding their effectiveness difficult. QUESTIONS/PURPOSES: The purpose of this study was to (1) determine if all reported periosteal procedures accelerate growth and increase the length of bones; (2) study the relative duration of these growth-accelerating effects at two time points; and (3) identify the periosteal procedure that results in the most growth. METHODS: Periosteal stripping (N = 8), periosteal transection (N = 8), periosteal resection (N = 8), (and) full periosteal release (N = 8) were performed on the tibiae of skeletally immature rabbits. Tibiae were collected 2 weeks postoperatively. The tibiae of additional cohorts of periosteal transection (N = 8), periosteal resection (N = 8), full periosteal release (N = 8), and repetitive periosteal transection (N = 8) were collected 8 weeks postoperatively. The contralateral tibiae served as an operative sham control in all cohorts. Fluorochrome bone labeling was used to measure growth rates, whereas high-resolution Faxitron imaging was performed to measure tibial lengths. Comparisons were then made between (1) experimental and sham controls; and (2) different procedures. Eight additional nonsurgical animals were included as age-matched controls. RESULTS: Growth (in microns) was accelerated at the proximal tibial physis on the tibia undergoing the periosteal surgical procedures versus the contralateral control limb after the transection (411 ± 27 versus 347 ± 18, p < 0.001 [mean ± SD]), resection (401 ± 33 versus 337 ± 31, p < 0.001), and full periosteal release (362 ± 45 versus 307 ± 33, p < 0.001), 2 weeks after the index procedure. Conversely, the periosteal stripping cohort trended toward less growth (344 ± 35) than the controls (356 ± 25; p = 0.08). No differences were found between limbs in the nonoperative controls. Tibial lengths for the experimental tibiae were longer at 2 weeks in the transection (1.6 ± 0.4 mm, p < 0.001), resection (1.6 ± 0.9 mm, p = 0.03), and full periosteal release (1.7 ± 0.5 mm, p < 0.001), whereas negligible differences were found between the tibiae of the nonoperative controls (0.13 ± 0.7 mm, p = 0.8) and stripping cohorts (0.10 ± 0.6 mm, p = 0.7). At 8 weeks, growth acceleration ceased at the proximal tibial physes in the transection cohort (174 ± 11 versus 176 ± 21, p = 0.8), and the control limbs actually grew faster than the experimental limbs after resection (194 ± 24 versus 178 ± 23, p = 0.02) and full periosteal release (193 ± 16 versus 175 ± 19, p < 0.01) cohorts. Growth rates were increased over control limbs, only in the repetitive transection cohort (190 ± 30 versus 169 ± 19, p = 0.01) at 8 weeks. Tibial lengths for the experimental tibiae remained longer at 8 weeks in the transection (1.4 ± 0.70 mm, p < 0.001), resection (2.2 ± 0.82 mm, p < 0.001), full periosteal release (1.6 ± 0.42 mm, p < 0.001), and repetitive periosteal transection (3.3 ± 1.1 mm, p < 0.001), whereas negligible differences were found between the tibiae of the nonoperative controls (-0.08 ± 0.58 mm, p = 0.8). Comparing the procedures at 2 weeks postoperatively, no differences were found in tibial lengths among the transection (2.1% ± 0.5% increase), resection (2.1% ± 1.1% increase), and full periosteal release (2.1% ± 0.6 %); however, all three demonstrated greater increased growth when compared with the stripping cohort (-0.10% ± 0.7%; p < 0.05). At 8 weeks no differences could be found between increased tibial lengths among the transection (1.5% ± 0.7%), resection (2.3% ± 0.9%), and full periosteal release (1.7% ± 0.4%). The repetitive transection produced the greatest over length increase (3.5% ± 1%), and this was greater than the acceleration generated by the single resection (p < 0.001) or the full periosteal release (p = 0.001). All four demonstrated an increase greater than the nonoperative control (0.09% ± 0.6%; p < 0.05). CONCLUSIONS: Transection of the longitudinally oriented periosteal fibers appears critical to accelerate growth in a rabbit model. CLINICAL RELEVANCE: These findings in an animal model support previous claims that limb overgrowth occurs as the result of periosteal disruption. Based on these findings in rabbits, we believe that less invasive procedures like periosteal transection are a promising avenue to explore in humans; clinical studies should seek to determine whether it is equally effective as more invasive procedures and its role as an adjunct to guided growth or distraction osteogenesis.

Effects of BMP-12-releasing sutures on Achilles tendon healing.

Tendon healing is a complex coordinated event orchestrated by numerous biologically active proteins. Unfortunately, tendons have limited regenerative potential and as a result, repair may be protracted months to years. Current treatment strategies do not offer localized delivery of biologically active proteins, which may result in reduced therapeutic efficacy. Surgical sutures coated with nanostructured minerals may provide a potentially universal tool to efficiently incorporate and deliver biologically active proteins directly to the wound. Additionally, previous reports indicated that treatment with bone morphogenetic protein-12 (BMP-12) improved tendon healing. Based on this information, we hypothesized that mineral-coated surgical sutures may be an effective platform for localized BMP-12 delivery to an injured tendon. The objective of this study was, therefore, to elucidate the healing effects of mineral-coated sutures releasing BMP-12 using a rat Achilles healing model. The effects of BMP-12-releasing sutures were also compared with standard BMP-12 delivery methods, including delivery of BMP-12 through collagen sponge or direct injection. Rat Achilles tendons were unilaterally transected and repaired using BMP-12-releasing suture (0, 0.15, 1.5, or 3.0 µg), collagen sponge (0 or 1.5 µg BMP-12), or direct injection (0 or 1.5 µg). By 14 days postinjury, repair with BMP-12-releasing sutures reduced the appearance of adhesions to the tendon and decreased total cell numbers. BMP-12 released from sutures and collagen sponge also tended to improve collagen organization when compared with BMP-12 delivered through injection. Based on these results, the release of a protein from sutures was able to elicit a biological response. Furthermore, BMP-12-releasing sutures modulated tendon healing, and the delivery method dictated the response of the healing tissue to BMP-12.

Interleukin-1 receptor antagonist modulates inflammation and scarring after ligament injury.

Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.

Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 10(6) or high dose 4 × 10(6) MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1ß, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.

Interleukin expression after injury and the effects of interleukin-1 receptor antagonist.

Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1ß, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery.