Kunihiko Hiramatsu

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.

SIK3 is essential for chondrocyte hypertrophy during skeletal development in mice

Chondrocyte hypertrophy is crucial for endochondral ossification, but the mechanism underlying this process is not fully understood. We report that salt-inducible kinase 3 (SIK3) deficiency causes severe inhibition of chondrocyte hypertrophy in mice. SIK3-deficient mice showed dwarfism as they aged, whereas body size was unaffected during embryogenesis. Anatomical and histological analyses revealed marked expansion of the growth plate and articular cartilage regions in the limbs, accumulation of chondrocytes in the sternum, ribs and spine, and impaired skull bone formation in SIK3-deficient mice. The primary phenotype in the skeletal tissue of SIK3-deficient mice was in the humerus at E14.5, where chondrocyte hypertrophy was markedly delayed. Chondrocyte hypertrophy was severely blocked until E18.5, and the proliferative chondrocytes occupied the inside of the humerus. Consistent with impaired chondrocyte hypertrophy in SIK3-deficient mice, native SIK3 expression was detected in the cytoplasm of prehypertrophic and hypertrophic chondrocytes in developing bones in embryos and in the growth plates in postnatal mice. HDAC4, a crucial repressor of chondrocyte hypertrophy, remained in the nuclei in SIK3-deficient chondrocytes, but was localized in the cytoplasm in wild-type hypertrophic chondrocytes. Molecular and cellular analyses demonstrated that SIK3 was required for anchoring HDAC4 in the cytoplasm, thereby releasing MEF2C, a crucial facilitator of chondrocyte hypertrophy, from suppression by HDAC4 in nuclei. Chondrocyte-specific overexpression of SIK3 induced closure of growth plates in adulthood, and the SIK3-deficient cartilage phenotype was rescued by transgenic SIK3 expression in the humerus. These results demonstrate an essential role for SIK3 in facilitating chondrocyte hypertrophy during skeletogenesis and growth plate maintenance.

Absorption of the Bone Fragment in Shoulders With Bony Bankart Lesions Caused by Recurrent Anterior Dislocations or Subluxations When Does It Occur

Background: Recently, bony defects of the glenoid in patients with traumatic anterior shoulder instability have been increasingly noticed. The bone fragment of a bony Bankart lesion is often utilized for Bankart repair, but the fragment is at times smaller than the glenoid defect. The reason for this mismatch in size is unknown. Hypothesis: The bone fragment of a bony Bankart lesion might gradually be absorbed over time. Study Design: Case series; Level of evidence, 4. Methods: A total of 163 shoulders were prospectively examined by computed tomography. In shoulders with bony Bankart lesions, glenoid defects and bone fragment absorption were assessed, and findings were compared with the time elapsed after the primary traumatic episode. When a bone fragment was not detected despite loss of the normal contour of the glenoid rim, the findings were classified as erosions if the rim appeared round and slightly compressed and classified as complete bone fragment absorption if the rim appeared straight and sharp. Results: There were no glenoid defects in 55 shoulders, erosions in 16 shoulders, and glenoid defects in 92 shoulders. The size of the glenoid defect was 0% to 10% in 15 shoulders, 10% to 20% in 44, 20% to 30% in 26, 30% to 40% in 6, and 40% to 50% in 1. The average defect size was 7.9% in shoulders scanned at <1 year, 10.7% between 1 and 2 years, and 11.3% at >2 years, indicating no relationship with time after trauma. Regarding bone fragment absorption, all 92 shoulders with glenoid defects showed absorption to some extent. The extent of absorption was <50% in 32 shoulders, >50% in 45, and 100% in 15. The average extent of absorption was 51.9% in shoulders scanned at <1 year, 65.3% between 1 and 2 years, and 70.0% at >2 years, indicating a significant relationship with time after trauma. Conclusion: Bone fragment absorption was seen in all of the shoulders with bony Bankart lesions. Most bone fragments showed severe absorption within 1 year after the primary traumatic episode. Before arthroscopic Bankart repair, not only glenoid defects but also bone fragment absorption should be assessed.

Low-intensity pulsed ultrasound increases bone ingrowth into porous hydroxyapatite ceramic

Synthetic porous ceramic made of hydroxyapatite (HA) has been used as a bone graft substitute. In the present study we investigated whether low-intensity pulsed ultrasound (LIPUS) accelerates bone ingrowth into the pores of HA ceramic. Application of LIPUS did not mechanically weaken porous ceramic that was immersed in water in vitro. In vivo experiments using rabbits showed that LIPUS application for 2 weeks significantly increased osteoblast number and bone area in the central part of the porous HA ceramic implanted in the femoral condyle in comparison with similarly implanted HA ceramic that was not exposed to LIPUS. LIPUS application for 3 weeks significantly increased mineralized tissue volume and mineral content in the porous HA ceramic. Wound healing assays revealed increased migration of MC3T3-E1 cells as a result of LIPUS treatment, partly accounting for the increased osteoblast number. Use of porous HA ceramic combined with LIPUS may be a promising treatment for filling large bone defects in a clinical setting.

Induction of chondrogenic cells from dermal fibroblast culture by defined factors does not involve a pluripotent state

There is a significant need for cell sources for cartilage regenerative medicine. It has been reported that the combined transduction of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) directly induces chondrogenic cells from mouse dermal fibroblast (MDF) culture. To gain insights into the process by which cellular characteristics are altered by transduction of c-Myc, Klf4 and SOX9, we examined marker gene expression in the MDF culture at various time points after transduction. The expression of fibroblast-markers was reduced first, followed by an increase in the expression of a chondrocyte-marker. We detected no expression of pluripotent markers at any time point examined. To determine whether or not induced chondrogenic cells go through a pluripotent state after transduction, we analyzed MDFs prepared from Nanog-GFP transgenic mice by monitoring expression of the GFP-labeled pluripotent marker Nanog-GFP in the MDF culture, using time-lapse microscopic observation. Whole-well time-lapse observation revealed that none of the induced chondrogenic cells displayed GFP fluorescence during induction. These results indicate that cells do not undergo a pluripotent state during direct induction of chondrogenic cells from fibroblast culture by transduction of c-Myc, Klf4 and SOX9.

Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo.

Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties through the forced expression of transcription factors. Herein, we investigated the in vitro hypertrophic differentiation and in vivo tissue formation capacity of induced chondrogenic cells (iChon cells) obtained through direct reprogramming. In vitro hypertrophic differentiation was detected in iChon cells that contained a doxycycline-inducible expression system for Klf4, cMyc, and Sox9. Furthermore, endochondral bone formation was detected after implantation in nude mice. The bone tissue was derived entirely from host origin, whereas cartilage tissue contained cells from both host and donor. The results obtained highlight the promise of cellular reprogramming for the creation of functional skeletal cells that can be used for novel bone healing strategies.

Expression of dominant negative TGF-β receptors inhibits cartilage formation in conditional transgenic mice

Although transforming growth factor-β (TGF-β) signaling has been implicated in cartilage formation in various studies, the exact role played by TGF-β in this process remains controversial. TGF-β signals are transduced through TGF-β type II receptor (TGF-βRΙΙ) and type I receptor (ALK5). Col2a1-Cre-mediated deletion of Tgfbr2 did not cause obvious defects in long bone formation, suggesting that TGF-β signals are dispensable for normal cartilage formation in the stage of condensing mesenchymal cells and chondrocytes or that related molecules can compensate for the lack of TGF-βRΙΙ. In the present study, we established a conditional transgenic mouse in which a dominant negative form of TGF-βRII (dnTGF-βRII) is expressed in condensing mesenchymal cells and chondrocytes in limbs using the Cre/loxP system. Recombination at loxP sites and expression of dnTgfbr2 were monitored by the disappearance of LacZ expression. The conditional transgenic mice expressing dnTgfbr2 developed hypoplastic cartilage. The phenotype was much more severe than that of Col2a1-Cre-mediated Tgfbr2 conditional knockout mice, although the pattern of dnTgfbr2 expression appears similar to the pattern of Tgfbr2 deletion. These phenotypic differences suggest that the signaling through TGF-β receptors is complex in cartilage.

Effects of suture site or penetration depth on anchor location in all-inside meniscal repair.

BACKGROUND To evaluate the effects of suture site or penetration depth on anchor location in all-inside meniscal repair. METHODS Eight fresh-frozen cadaveric knees were evaluated after meniscal repair using eight FasT-Fix360 (FF360) devices (Smith & Nephew Endoscopy, Andover, MA) (16 anchors) for each knee. The penetration depth was 14mm, the distance same from the periphery to insertion point, in four knees (Group A) and that in the remaining four knees (Group B) was 18mm. The anchor location in two groups was evaluated after attentive dissection. RESULTS Of 32 anchors for the medial meniscus, 94% were on the capsule, including the superficial medial collateral ligament (sMCL) in both groups. For the lateral meniscus, 47% anchors in Group A and 44% anchors in Group B were on the capsule. Total three anchors were over the lateral collateral ligament (LCL), whereas 15 anchors were behind the popliteus tendon (POP). Although all three anchors settled in the subcutaneous fat were in Group B, no significant difference was observed in anchor location between two groups. CONCLUSIONS Secure fixation to thin membranous tissue can be achieved for the medial meniscal repair using FF360, while some were located in/on bunchy LCL or POP in lateral meniscal repair. Only anchors with additional four-millimeter penetration depth were in the subcutaneous fat, although there was no effect of the penetration depth to anchor location. Clinically, for lateral meniscal repair, penetrating toward POP/LCL should be avoided and four-millimeter deeper penetration depth might be a risk for the subcutaneous irritation.

Ultrasonographic Evaluation of the Early Healing Process After Achilles Tendon Repair

Background: Little is known about early healing of repaired Achilles tendons on imaging, particularly up to 6 months postoperatively, when patients generally return to participation in sports. Purpose: To examine changes in repaired Achilles tendon healing with ultrasonography for up to 12 months after surgery. Study Design: Case series; Level of evidence, 4. Methods: Ultrasonographic images of 26 ruptured Achilles tendons were analyzed at 1, 2, 3, 4, 6, and 12 months after primary repair. The cross-sectional areas (CSAs) and intratendinous morphology of the repaired tendons were evaluated using the authors’ own grading system (tendon repair scores), which assessed the anechoic tendon defect area, intratendinous hyperechoic area, continuity of intratendinous fibrillar appearance, and paratendinous edema. Results: The mean ratios (%) of the CSA for the affected versus unaffected side of repaired Achilles tendons gradually increased postoperatively, reached a maximum (632%) at 6 months, and then decreased at 12 months. The mean tendon repair scores increased over time and reached a plateau at 6 months. Conclusion: Ultrasonography is useful to observe the intratendinous morphology of repaired Achilles tendons and to provide useful information for patients who wish to return to sports. Clinical parameters such as strength, functional performance, and quality of healed repaired tendons should also be assessed before allowing patients to return to sports.