Mark L. Wang

Characterization of Multipotential Mesenchymal Progenitor Cells Derived from Human Trabecular Bone

The in vitro culture of human trabecular bone‐derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase‐treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone‐derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73+, STRO‐1+, CD105+, CD34−, CD45−, CD144− cell population resident within collagenase‐treated, culture‐processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long‐term culture expansion. When cultured as described, the trabecular bone‐derived cells display stem cell‐like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long‐term in vitro culture.

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells

Long‐term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS‐treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi‐treated hMSC cultures revealed patchy groups of cells displaying disorganized cyto‐skeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.

Direct and indirect induction of apoptosis in human mesenchymal stem cells in response to titanium particles.

The most frequent complication of total joint arthroplasty is periprosthetic osteolysis initiated by an inflammatory response to orthopaedic wear debris, which if left untreated, can result in implant instability and failure, eventually requiring revision surgery. We have previously reported that osteogenic differentiation of human marrow stroma‐derived mesenchymal stem cells (hMSCs) is suppressed upon exposure to titanium particles, accompanied by reduced bone sialoprotein (BSP) gene expression, diminished production of collagen type I and BSP, decreased cellular viability and proliferation, and inhibition of extracellular matrix mineralization. In this study, we have further investigated hMSC cytotoxicity upon exposure to submicron particles of commercially pure titanium (cpTi) and zirconium oxide (ZrO2). Our results showed that direct exposure to cpTi and ZrO2 particles compromises cell viability through the induction of apoptosis, eliciting increased levels of the tumor suppressor proteins p53 and p73, in a manner dependent on material composition, particle dosage, and time. Additionally, conditioned medium collected from hMSCs exposed to cpTi particles, but not to ZrO2 particles, is cytotoxic to hMSCs, inducing apoptosis in the absence of particles. These findings demonstrate that exposure to orthopaedically derived wear particles can compromise hMSC viability through the direct and indirect induction of apoptosis. Thus, prolonged in vivo exposure of marrow‐derived hMSCs to implant‐derived wear debris is likely to reduce the population of viable osteoprogenitor cells, and may contribute to poor periprosthetic bone quality and implant loosening.

A Simple, High-Yield Method for Obtaining Multipotential Mesenchymal Progenitor Cells from Trabecular Bone

In vitro cultures of primary, human trabecular bone-derived cells represent a useful system for investigation of the biology of osteoblasts. Our recent discovery of the multilineage mesenchymal differentiation potential of trabecular bone-derived cells suggests the potential application of these cells as mesenchymal progenitors for tissue repair and regeneration. Such applications are crucially dependent on efficient cellisolation protocols to yield cells that optimally proliferate and differentiate. In this study, we describe a simple, high-yield procedure, requiring minimal culture expansion, for the isolation of mesenchymal progenitor cells from human trabecular bone. Moreover, these cells retain their ability to differentiate along multiple mesenchymal lineages through successive subculturing. Cell populations isolated and cultured as described here allow the efficient acquisition of a clinically significant number of cells, which may be used as the cell source for tissue-engineering applications.

TGF-β1 calcium signaling increases α5 integrin expression in osteoblasts

TGF‐β1 is a potent osteoactive factor and exhibits a wide variety of effects on osteoblasts, most of which are mediated through receptor associated Smad proteins. We have recently reported a novel TGF‐β1 intracellular Ca2+ signaling pathway in osteoblasts, and found that this signaling is required for the TGF‐β1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and α5β1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF‐β1 stimulated Ca2+ signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF‐β1 induced intracellular Ca2+ signal is responsible, in part, for the stimulation of expression of α5 integrin, but not of β1 integrin or fibronectin. Increased levels of α5 integrin protein and mRNA were seen as early as 12 h after TGF‐β1 treatment, but were inhibited by co‐treatment of cells with nifedipine, a selective L‐type Ca2+ channel blocker. TGF‐β1 treatment increased both fibronectin and β1 integrin protein production within 48 h, in a manner unaffected by co‐treatment with nifedipine.

TGF-β1-Stimulated Osteoblasts Require Intracellular Calcium Signaling for Enhanced α5 Integrin Expression

Abstract: The osteoactive factor, transforming growth factor β1 (TGF‐β1), influences osteoblast activity and bone function. We recently characterized a Smad‐independent TGF‐β1‐induced Ca2+ signal in human osteoblasts (HOB) and demonstrated its importance in cell adhesion. Here, we further elucidate the role of the TGF‐β1 Ca2+ signal in the mechanics of HOB adhesion. Osteoblast interaction with fibronectin (FN) through α5β1 integrin is principally responsible for osteoblast‐substrate adhesion. Our results show that the TGF‐β1 intracellular Ca2+ signal is responsible, in part, for stimulation of α5 integrin expression, but not β1 integrin or FN expression. Increased α5 integrin protein and mRNA expression was seen as early as 12 h after TGF‐β1 treatment, but was inhibited by cotreatment with nifedipine, a Ca2+ channel blocker. TGF‐β1 increased both FN and β1 integrin protein production within 48 h, independent of nifedipine cotreatment. Immunofluorescence observations revealed that TGF‐β1 increased α5 integrin staining, clustering, and colocalization with the actin cytoskeleton, effects that were blocked by nifedipine. The TGF‐β1 Ca2+ signal, a pathway crucial for HOB adhesion, enhances α5 integrin expression, focal contact formation, and cytoskeleton reorganization. These early events are necessary for osteoblast adhesion; thus they determine the fate of the cell and ultimately affect bone function.

Auxiliary proteins that facilitate formation of collagen‐rich deposits in the posterior knee capsule in a rabbit‐based joint contracture model

Post‐traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen‐rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α‐subunit of prolyl 4‐hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril‐stabilizing cross‐links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti‐fibrotic targets.

Lunatocapitate and Triquetrohamate Arthrodeses for Degenerative Arthritis of the Wrist

PURPOSE Proximal row carpectomy and 4-corner arthrodesis are 2 well-established motion-preserving treatment strategies for scapholunate advanced collapse. In this study, we present an arthrodesis technique involving the capitolunate and triquetrohamate joints as another potential treatment option. METHODS From 2000 to 2009, 27 consecutive patients with degenerative scapholunate advanced collapse and scaphoid nonunion advanced collapse were evaluated prospectively and treated with scaphoid excision and intercarpal arthrodesis between the capitate and lunate and between the hamate and triquetrum. This cohort consisted of 18 men and 9 women, involving dominant-sided surgery in 20 of 27 patients. Two patients were active smokers, and 3 cases were work related. Average age at time of surgery was 55 ± 3 years, and average follow-up was 51 ± 7 months. Preoperative and postoperative range of motion, grip strength, and radiographic evidence of osseous union were documented. Standardized Patient-Rated Wrist Evaluation scores for both pain and function were collected. RESULTS Wrist extension and flexion were decreased after surgery by 17% and 25% respectively, yielding a 21% decrease in mean flexion-extension arc. There was no significant difference with regard to postoperative radial and ulnar deviation or mean coronal plane arc compared to preoperative values. Compared to the contralateral side, preoperative and postoperative grip strength were 53% and 70%, respectively. The average operative-sided grip strength increased by 27%. The mean Patient-Rated Wrist Evaluation pain score was 11 ± 3 (of 50). The mean Patient-Rated Wrist Evaluation functional score was 17 ± 5 (of 100). Complications included 1 nonunion (yielding a 96% fusion rate), 1 median neuropathy (which resolved), and 2 superficial wound infections (treated successfully with oral antibiotics). CONCLUSIONS Arthrodesis of the capitolunate and triquetrohamate joints offers a motion-preserving strategy with a high union rate and good clinical function and pain outcomes for the treatment for scapholunate advanced collapse and scaphoid nonunion advanced collapse. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic IV.

Postsurgical bleeding following treatment of a chronic Morel-Lavallée lesion.

Morel-Lavallée lesions are closed soft-tissue degloving injuries that occur when the skin and subcutaneous tissues are separated from underlying fascia as a result of a sudden shearing force. The space that is created has the potential to be filled with a mixture of blood, lymph fluid, or necrotic fat, which may easily become infected. The optimal treatment of Morel-Lavallée lesions is controversial. We present a case of a 29-year-old man who sustained pelvic fractures in a motorcycle accident and 1 year later presented with a painless, slowly expanding left thigh mass. Treatment, consisting of open debridement and primary closure, was complicated by significant postoperative bleeding requiring reoperation. The patient was followed clinically as an outpatient, and at 6-month follow-up he was doing well and had no evidence of recurrence of the lesion. Morel-Lavallée lesions are rare complications that are often missed or underappreciated at the time of initial injury. In many cases these lesions can persist for months before they are recognized. For long-standing Morel-Lavallée lesions, it is important to determine the nature of the fluid in the cavity before planning treatment. In the current case, it was unclear based on the official radiographic interpretation whether the lesion was filled with frank blood versus serous or serosanguineous fluid.

Blocking collagen fibril formation in injured knees reduces flexion contracture in a rabbit model.

Post‐traumatic joint contracture is a frequent orthopaedic complication that limits the movement of injured joints, thereby severely impairing affected patients. Non‐surgical and surgical treatments for joint contracture often fail to improve the range of motion. In this study, we tested a hypothesis that limiting the formation of collagen‐rich tissue in the capsules of injured joints would reduce the consequences of the fibrotic response and improve joint mobility. We targeted the formation of collagen fibrils, the main component of fibrotic deposits formed within the tissues of injured joints, by employing a relevant rabbit model to test the utility of a custom‐engineered antibody. The antibody was delivered directly to the cavities of injured knees in order to block the formation of collagen fibrils produced in response to injury. In comparison to the non‐treated control, mechanical tests of the antibody‐treated knees demonstrated a significant reduction of flexion contracture. Detailed microscopic and biochemical studies verified that this reduction resulted from the antibody‐mediated blocking of the assembly of collagen fibrils. These findings indicate that extracellular processes associated with excessive formation of fibrotic tissue represent a valid target for limiting post‐traumatic joint stiffness.