Bone marrow-derived cells from the footprint infiltrate into the repaired rotator cuff.

BACKGROUND: Cells from the bone marrow are considered important during the rotator cuff repair process, but the kinetics of bone marrow-derived cells in this process is unknown. PURPOSE: To analyze the kinetics of bone marrow cells during the rotator cuff repair process, to review whether or not they are histologically involved in rotator cuff healing, and to analyze the biomechanics of the repaired tissues. METHODS: Bone marrow chimeric rats that express green fluorescent protein (GFP) only in bone marrow- and circulation-derived cells were created. Bilateral supraspinatus tendons were separated from the greater tuberosity of the humeral head to produce a rotator cuff transection model. Drilling into the bone marrow was performed in the greater tuberosity of the right humerus and the supraspinatus tendon was repaired (drilling group), while the supraspinatus tendon was repaired on the left shoulder without drilling (control group). We examined the histology of the rotator cuff, the ultimate force-to-failure, and the proportion of GFP-positive cells in the repaired rotator cuff at 2, 4 and 8 weeks after surgery. RESULTS: Mesenchymal cells were observed in the repaired rotator cuff at 2 weeks in both groups. There were more GFP-positive cells in the drilling group than the control group at 2, 4 and 8 weeks. The ultimate force-to-failure was significantly higher in the drilling group than the control group at 4 and 8 weeks. CONCLUSION: Bone marrow-derived cells passed through holes drilled in the humerus footprint, infiltrated the repaired rotator cuff and contributed to postsurgical rotator cuff healing.

The fate and role of bone graft-derived cells after autologous tendon and bone transplantation into the bone tunnel.

BACKGROUND: Grafting bone between the tendon graft and the bone tunnel in anterior cruciate ligament reconstruction increases the mechanical strength of the tendon graft. However, the biological role of the bone graft is unclear. The purpose of this research was to elucidate the role of bone graft cells after autologous tendon graft into the bone tunnel with an autologous bone graft in green fluorescent protein (GFP) transgenic rats. METHODS: The Achilles tendons of Sprague-Dawley (SD) wild-type rats and bone of GFP rats were harvested and transplanted into bone tunnels drilled in the femurs at the knees of SD rats. The femurs were harvested at 1, 2, and 4 weeks after transplantation and histologically investigated using hematoxylin and eosin staining and immunostaining of heat shock protein 47 (HSP47), macrophages, and type I and type III collagens. Biomechanical tests were performed on the tendon graft 2 and 4 weeks after transplantation to evaluate the ultimate force to failure. RESULTS: A small number of GFP-positive cells was seen in the tendon graft 2 weeks after transplantation. The cell count in the tendon graft was increased at 4 weeks after transplantation. HSP47-positive cells and macrophage-stained cells present in the tendon graft corresponded with the GFP-positive cells. By 2 weeks after transplantation, the relative areas of immunostained type I and III collagens in the tendon graft had declined significantly in the bone graft group compared to the control. The ultimate failure load in the bone graft group was higher than that in the control group at both 2 and 4 weeks after transplantation. CONCLUSIONS: This research showed that, within 4 weeks of transplantation, bone graft cells migrate to the tendon graft, where they differentiate into cells involved in collagen production and macrophages. Bone graft cells may contribute to the early stage remodeling of tendon grafts.

Immunohistochemical analysis of the effects of estrogen on intraarticular neurogenic inflammation in a rat anterior cruciate ligament transection model of osteoarthritis.

Synovitis is considered as one of the factors associated with the pathogenesis of osteoarthritis (OA). There is currently a significant amount of research linking estrogen deficiencies with the development of OA in estrogen-deficient women, including postmenopausal women; however, the exact etiology remains unclear. Various neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), have been shown to contribute to synovitis in OA joints, and the influence of estrogen on the expressions of SP and CGRP in the synovium of OA joints has been noted. After ovariectomy (OVX) followed by estradiol (E2) replacement, 24 female rats were divided into three groups: OVX group, OVX + E2 replacement group (E2 group), and a sham group. All rats underwent transection of the anterior cruciate ligament at the same time. After 30 days, the histological findings of knee joints by hematoxylin-eosin staining and immunofluorescence staining of protein gene product 9.5 (pan-neuronal marker), SP, and CGRP were compared among experimental groups. The degree of synovitis in the OVX group was higher than in the E2 and sham groups. No significant differences in the density of protein gene product 9.5-immunoreactive nerve fibers were observed among the three experimental groups, but the density of SP- or CGRP-immunoreactive nerve fibers in the OVX group was significantly higher than in the E2 and sham groups. These findings suggest that estrogen partly regulates intraarticular neurogenic inflammation in OA joints by modulating the expressions of neuropeptides in the synovium.

Characteristics of donor and host cells in the early remodeling process after transplant of Achilles tendon with and without live cells for the treatment of rotator cuff defect--what is the ideal graft for the treatment of massive rotator cuff defects?

PURPOSE: We examined the characteristics of donor and host cells in the early remodeling process after transplant of Achilles tendon with and without live cells to repair rotator cuff defects. We also clarified which graft with or without live cells was superior in the early remodeling process. MATERIALS AND METHODS: Sprague-Dawley (SD) rats and green fluorescent protein (GFP) rats were used; they were divided into 3 groups: in group SD, the Achilles tendons of GFP rats were transplanted into the defects of SD rats; in group GFP, the Achilles tendons of SD rats were transplanted into GFP rats; in group GFP-Fr, frozen Achilles tendons of SD rats were transplanted into GFP rats. At 3 and 7 days after surgery, these sections were examined histologically and immunohistochemically with anti-heat shock protein (HSP) 47 and anti-macrophage antibodies. RESULTS: Donor cells gradually decreased, but HSP47-positive donor cells were detected at 3 days in group SD. Host cells infiltrated into the graft from the surrounding tissue, and their numbers in groups SD and GFP gradually increased more significantly than in group GFP-Fr. Macrophages derived from the donor tissue were absent in all groups. The remodeling process of the frozen graft was slower than that in the case of the graft that was not frozen. CONCLUSION: These results demonstrate that live donor cells have a positive effect on the remodeling process. Therefore, autografts with live cells considered to be preferred to frozen allografts or synthetic materials without live cells for transplant for rotator cuff defects.

In vivo effects of ovarian steroid hormones on the expressions of estrogen receptors and the composition of extracellular matrix in the anterior cruciate ligament in rats.

Female athletes have a significantly higher rate of anterior cruciate ligament (ACL) injury than their male counterparts. Sex steroid hormones are considered to have an influence as risk factors for female ACL injuries. We hypothesized that estrogen and progesterone have specific and synergistic influences on the composition of extracellular matrix in ACL. By ovariectomy (OVX) followed by subcutaneous estradiol (E2) and/or progesterone (P4) replacement, 40 female rats were divided into 5 groups: E2, P4, combined E2 and P4 (EP), OVX control, and sham group. After 30 days, using undecalcified sections of knee joints in conjunction with immunofluorescence staining of estrogen receptor alpha and beta (ERalpha and ERbeta), collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP), the immunoreactivities of these proteins in two distinct parts of ACL, proximal and middle portions, were compared semiquantitatively among experimental groups. By E2 replacement, the expressions of ERalpha in ACL fibroblasts were elevated compared to the OVX group. At the proximal portion, the immunoreactivities of type 1 collagen by E2 replacement, type 3 collagen by P4 replacement, and COMP by E2 or P4 replacement were significantly reduced. At the middle portion, the immunoreactivity of type 3 collagen was significantly elevated by E2 replacement. However, no differences were observed between the sham and OVX groups. These findings suggest that ACL is ER-dependent and that ovarian hormones alter ligament tissue composition, especially at the proximal portion. Female hormonal influences are partly involved in the biological properties of ACL.

Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.

Circulation-derived cells play a crucial role in the healing processes of tissue. In early phases of tendon healing processes, circulation-derived cells temporarily exist in the wounded area to initiate the healing process and decrease in number with time. We assumed that a delay of time-dependent decrease in circulation-derived cells could improve the healing of tendons. In this study, we injected platelet-rich plasma (PRP) containing various kinds of growth factors into the wounded area of the patellar tendon, and compared the effects on activation of circulation-derived cells and enhancement of tendon healing with a control group (no PRP injection). To follow the circulation-derived cells, we used a green fluorescent protein (GFP) chimeric rat expressing GFP in the circulating cells and bone marrow cells. In the PRP group, the numbers of GFP-positive cells and heat-shock protein (HSP47; collagen-specific molecular chaperone)-positive cells were significantly higher than in the control group at 3 and 7 days after injury. At the same time, the immunoreactivity for types I and III collagen was higher in the PRP group than in the control group at early phase of tendon healing. These findings suggest that locally injected PRP is useful as an activator of circulation-derived cells for enhancement of the initial tendon healing process.

Behavior of host and graft cells in the early remodeling process of rotator cuff defects in a transgenic animal model.

Autologous tissue graft is one of the treatment options for a large rotator cuff defect. To develop appropriate strategies for enhanced solid graft integration at the bone-tendon interface and tendon-tendon interface, clarifying the fate of the graft and host cells that contribute to repair and remodeling is necessary. We have developed a new grafting model using green fluorescent protein-transgenic rats and wild-type rats to simulate autologous transplantation for examining the behavior of the host and graft cells in the remodeling process after tendon grafting. We found that the host cells commenced proliferation in the graft at 1 day after grafting. The host cells infiltrated into the graft from the subacromial synovium, proximal tendon, and bone-tendon insertion. The number of graft-derived cells decreased with time. Our result clearly demonstrated that host cells, rather than graft cells, were essential for rotator cuff remodeling after tendon grafting for rotator cuff defect.

Localization of estrogen receptors alpha and beta in the articular surface of the rat femur.

It has been suggested that the degradation of the articular cartilage and osteoarthritis (OA) are associated with gender and the estrogen hormone. Although many investigators have reported the presence of the estrogen receptors (ERs) alpha and beta in the articular cartilage, the localization of these receptors and the difference in their in vivo expression have not yet been clearly demonstrated. We performed immunofluorescence staining of ERalpha and ERbeta to elucidate the localization of the ERs and to note the effects of gender and the aging process on these receptors. The results revealed that ERalpha and ERbeta were expressed in the articular cartilage and subchondral bone layers of adult rats of both sexes. We also observed the high expression of these receptors in immature rats. In contrast, their expression levels decreased in an ovariectomised model, as a simulation of postmenopause, and in aged female rats. Therefore, this study suggests the direct effects of estrogen and ER expression on articular surface metabolism.

GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing.

The healing of an injured musculoskeletal system requires an influx of mesenchymal cells that can differentiate into osteoblasts, fibroblasts, chondroblasts, and skeletal myoblasts. However, whether these mesenchymal cells arise from the circulation (bone marrow) or the injured tissues themselves has been controversial. To reveal the spatiotemporal characteristics of the reparative mesenchymal cells, we investigated the healing process after patellar tendon injury using two types of green fluorescent protein (GFP) chimeric rats; one expressing GFP in the circulating cells, and the other expressing it in the patellar tendon. We analyzed the behavior of GFP-positive cells after experimental tendon injury in both chimeric rats to clarify the origin of reparative cells. At 24 h after the injury, the wound contained circulation-derived cells but not tendon-derived cells. Tendon-derived cells first appeared in the wounded area at 3 days after the injury, and had significantly increased in number with time and had maintained a high level of proliferative activity until 7 days after the injury, whereas the circulation-derived cells had decreased in number and had been replaced by the tendon-derived cells. These findings suggest that circulation-derived and tendon-derived cells contribute to the healing of tendons in different periods as part of a biphasic process.