De Novo Reconstruction of a Hybrid Patella by Staged Fabrication of a Microvascular Bone Transplant with an Osteointegrated Prosthetic Socket.

Total patellectomy is sometimes unavoidable but usually results in severely impaired function, pain, and instability in the affected knee. Any patellar prosthetic solutions rely on a certain amount of remaining bone and therefore are not applicable after total patellectomy. Traditionally, reconstruction of a neopatella by avascular or allogeneic bone grafts is hampered by mechanical failure, resorption, or infection. We developed a new, 3-stage approach to reconstruct a hybrid patella composed of a revascularized scapula tip transplant fabricated with a prosthetic socket. The procedure is safe and provides optimal healing and prosthetic osteointegration through viable bone and dynamic stability to the considerable load a patella has to bear in unrestricted mobility. The technique also demonstrates successful integration of orthopedic prosthetic devices into current flap fabrication concepts.

Non-Hematopoietic Essential Functions of Bone Marrow Cells: A Review of Scientific and Clinical Literature and Rationale for Treating Bone Defects.

Hematopoiesis as the only essential function of bone marrow cells has been challenged for several decades through basic science (in vitro and in vivo) and clinical data. Such work has shed light on two other essential functions of bone marrow cells: osteopoiesis and angio-genesis/vasculogenesis. Clinical utility of autologous concentrated bone marrow aspirate (CBMA) has demonstrated both safety and efficacy in treating bone defects. Moreover, CBMA has been shown to be comparable to the gold standard of iliac crest bone graft (ICBG), or autograft, with regard to being osteogenic and osteoinductive. ICBG is not considered an advanced therapy medicinal product (ATMP), but CBMA may become regulated as an ATMP. The European Medicines Agency Committee for Advanced Therapies (EMA:CAT) has issued a reflection paper (20 June 2014) in which reversal of the 2013 ruling that CBMA is a non-ATMP has been proposed. We review bone marrow cell involvement in osteopoiesis and angiogenesis/vasculogenesis to examine EMA:CAT 2013 decision to use CBMA for treatment of osteonecrosis (e.g, of the femoral head) should be considered a non-ATMP. This paper is intended to provide discussion on the 20 June 2014 reflection paper by reviewing two non-hematopoietic essential functions of bone marrow cells. Additionally, we provide clinical and scientific rationale for treating osteonecrosis with CBMA.

Bridging the gap: bone marrow aspiration concentrate reduces autologous bone grafting in osseous defects.

Although autologous bone grafting represents an effective tool to induce osteogenic regeneration in local bone defects or pseudarthroses, it is associated with significant donor site morbidity and limited by the amount available for grafting. We investigate the potency of bone marrow aspiration concentrate (BMAC) to augment bone grafting and support bone healing. The functional and radiographic outcome of 39 patients with volumetric bone deficiencies treated with BMAC are presented and evaluated in a prospective clinical trial. A collagen sponge (Col) served as scaffold in 12 patients and a bovine hydroxyapatite (HA) was applied in the other 27 individuals. The minimal follow-up was 6 months. Clinical and radiographic findings were completed by in vitro data. All patients showed new bone formation in radiographs during follow-up. However, two patients underwent revision surgery due to a lack in bone healing. In contrast to the Col group, the postoperative bone formation appeared earlier in the HA group (HA group: 6.8 weeks vs. Col group 13.6 weeks). Complete bone healing was achieved in the HA group after 17.3 weeks compared to 22.4 weeks in the Col group. The average concentration factor of BMAC was 5.2 (SD 1.3). Flow cytometry confirmed the mesenchymal nature of the cells. Cells from BMAC created earlier and larger colonies of forming units fibroblasts (CFU-F) compared to cells from bone marrow aspirate. BMAC combined with HA can reduce the time needed for healing of bone defects when compared to BMAC in combination with collagen sponge.

The Daily Activity Questionnaire: a novel questionnaire to assess patient activity after total hip arthroplasty.

Expensive electronic accelerometers are the only validated method to determine patient activity levels. The aim of this study was to develop a clinical questionnaire to assess patient activity. The Daily Activity Questionnaire (DAQ) was developed and evaluated using 3 groups of patients with osteoarthritis of the hip. A total of 160 patients underwent 855 days of monitoring. Practicability, reliability, and validity of the new questionnaire were assessed. The test-retest reliability of the DAQ was comparable to the electronic accelerometer StepWatch (ICC = 0.77-0.89). A significant correlation between the DAQ and the StepWatch was found (r = 0.742). The DAQ is a reliable and valid instrument to measure patient activity.

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer.

INTRODUCTION: The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures. METHODS: Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 x 10(2) viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. RESULTS: Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size. CONCLUSIONS: BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.

Patient activity after total hip arthroplasty declines with advancing age.

Evaluation of patient activity is essential for clinical decision making before THA. To correlate age progression to patient activity after THA, we determined the number of walking cycles of 105 patients in different age groups by decades. Patients on average performed 6144 walking cycles per day (2.24 million cycles per year). Men were more active than women. The highest activity occurred in patients between 50 and 59 years of age, with a constant decrease in activity with advancing age. However, within age groups, we observed up to sixfold differences in the number of walking cycles per day. In addition to declining activity with advancing age, higher body mass index correlated with lower step counts. The high mean measured number of walking cycles, which were even higher than those reported for subjects without an arthroplasty, suggests patients benefit from THA. Female gender, advanced age, and obesity correlated with lower activity. Owing to the high intragroup variability of our results, preoperative evaluation of patient activity levels, individual patient factors, and patient demands, should be considered in clinical practice.

Safety of autologous bone marrow aspiration concentrate transplantation: initial experiences in 101 patients.

The clinical application of cellular based therapies with ex vivo cultivation for the treatment of diseases of the musculoskeletal system has until now been limited. In particular, the advanced laboratory and technical effort necessary, regulatory issues as well as high costs are major obstacles. On the other hand, newly developed cell therapy systems permit intra-operative enrichment and application of mesenchymal and progenitor stem cells from bone marrow aspirate concentrate (BMAC) in one single operative session. The objective of the present clinical surveillance study was to evaluate new bone formation after the application of BMAC as well as to record any possible therapy-specific complicationsFor this purpose, the clinical-radiological progress of a total of 101 patients with various bone healing disturbances was documented (surveillance study). The study included 37 necrosis of the head of the femur, 32 avascular necroses/bone marrow edema of other localization, 12 non-unions, 20 other defects. The application of BMAC was performed in the presence of osteonecrosis via a local injection as part of a core decompression (n=72) or by the local adsorption of intra-operative cellular bone substitution material (scaffold) incubated with BMAC during osteosynthesis (n=17) or in further surgery (n=12).After an average of 14 months (2-24 months), the patients were re-examined clinically and radiologically and interviewed. Further surgery was necessary in 2 patients within the follow-up period. These were due to a progression of a collapsed head of the femur with initial necrosis in ARCO Stage III, as well as inadequate new bone formation with secondary loss of correction after periprosthetic femoral fracture. The latter healed after repeated osteosynthesis plus BMAC application without any consequences. Other than these 2 patients, no further complications were observed. In particular, no infections, no excessive new bone formation, no induction of tumor formation, as well as no morbidity due to the bone marrow aspiration from the iliac crest were seen.There were no specific complications within the short follow-up period and a simple intra-operative use of the system for different forms of bone loss could be demonstrated. In the authors' opinion, the on-site preparation of the bone marrow cells within the operating theater eliminates the specific risk of ex vivo cell proliferation and has a safety advantage in the use of autologous cell therapy for bone regeneration. Additional studies should be completed to determine efficacy.

In vitro investigation of orthopedic titanium-coated and brushite-coated surfaces using human osteoblasts in the presence of gentamycin.

Anti-infective coatings have been developed to protect the surfaces of cementless implants from bacterial colonization that is known to be a prerequisite for device-related infection. The aim of this study is to investigate the effect of brushite-coated arthroplasty surfaces on human osteoblasts and to evaluate the impact of concomitant exposure to gentamycin. We cultured human osteoblasts (hFOB 1.19) on brushite-coated and uncoated titanium alloy in the presence of gentamycin and analyzed cell function and vitality. Our results show that brushite-coated titanium alloy surfaces supported the function of osteoblasts and the expression of extracellular matrix even in the presence of highly dosed gentamycin. Brushite-coated titanium alloy surfaces supported osteogenic function, indicating that this coating could enhance implant osteointegration in vivo. Concomitant exposure to gentamycin slightly decreased osteoblastic activity in vitro, suggesting that there might also be negative effects in vivo. However, in vivo studies are necessary to validate these in vitro findings.

9- to 11-year results of cemented titanium mueller straight stem in total hip arthroplasty.

This retrospective study reviewed 9- to 11-year results after total hip arthroplasty (THA) with cemented titanium stems (Mueller-Straight-Stem). Ninety-one patients (110 hips) were examined clinically and radiologically at an average 9.5-year follow-up. Revisions for aseptic loosening were performed in 4 (4%) patients. Subsidence or varus position could only be observed in one of these patients. Radiolucent lines were found in 37 patients, mainly located around the proximal zones of the stem (zone 1, 7, 8, and 14). Harris scores were good or excellent in 78% and satisfactory in 20% of patients. The 9.5-year survival rate of the cemented titanium stem with regard to aseptic loosening was 96.4%. Body weight was significantly higher (88 +/- 5.4 kg) in the 4 patients with aseptic loosening, compared to patients without radiolucent lines (75 +/- 15 kg). The body weight to stem surface ratio showed a significant difference (1.5 kg/cm2 versus 1 kg/cm2; P < .05). No significant differences were found in other factors, including sex, size or type of stem, Harris score, heterotopic ossification, or body mass index. Good long-term results can be achieved with cemented titanium stem implants. This titanium implant is recommended for patients with hypersensitivity to chrome, cobalt, and nickel. mplanting the biggest possible stem seems to be most beneficial.

No migration of acetabular cups after prophylaxis for heterotopic ossification.

The possibility of migration of cementless cups in total joints after prophylaxis of heterotopic ossification with irradiation or nonsteroidal antiinflammatory drugs is a concern. Data investigating component stability with digital methods are lacking. This prospective study analyzed the migration of cementless cups after indomethacin and irradiation prophylaxis with the digital Einzel-Bild-Röntgen-Analyse tool. The irradiation group (106 hips) and the indomethacin group (98 hips) were compared with 82 hips that did not receive any prophylaxis. The same cementless acetabular implants were used in all cases, and patients were observed clinically and radiographically at 2 and 5 years. At the 5-year followup, the number of cups that showed migration greater than 1 mm in the irradiation group (five), the indomethacin group (three), and the control group (four) were not different. No cup was considered loose on the radiographs and no patient underwent revision surgery. The results of our study indicate irradiation or short-course use of indomethacin for prophylaxis of heterotopic ossification did not negatively affect the stability of cementless cups in primary total hip arthroplasties.

Minimally invasive anterolateral approach to the hip: risk to the superior gluteal nerve.

BACKGROUND: Minimally invasive approaches to the hip show promise of less muscle trauma compared to conventional approaches. What is the risk of damage to the superior gluteal nerve? We studied the course of the superior gluteal nerve. METHOD: 20 legs of 11 formalin-fixed Caucasian cadavers were dissected and the course and the distances of the superior gluteal nerve branches from the tip of the greater trochanter were documented. RESULTS: The branch of the gluteal superior nerve leading to the gluteal minimus muscle was 33 (20-50) mm from the tip of the greater trochanter, within a deeper layer. The nearest point of the superior gluteal nerve branches from the tip of the greater trochanter in the posterior region was 19 (10-30) mm, in the middle region 20 (20-30) mm and in the anterior region 20 (10-35) mm. In half of the cases, a distal intermuscular branch between gluteal medius and tensor fasciae latae muscle could be found, mean 27 (10-40) mm caudal and 38 (25-60) mm ventral to the tip of the greater trochanter. This distal branch is considered to create a loop with upper branches of the superior gluteal nerve within the tensor fasciae muscle. INTERPRETATION: The safe zone for the superior gluteal nerve was smaller than previously reported. Use of a minimal direct lateral approach puts the inferior branches within the gluteal medius at risk; however, a minimal anterolateral approach to the hip may compromise branches of the superior gluteal nerve to the tensor fasciae latae muscle.

Effect of polyhexanide and gentamycin on human osteoblasts and endothelial cells.

QUESTIONS UNDER STUDY: Infection of total joint replacements is painful, disabling and difficult to treat because of the increasing bacterial resistance against antibiotics. In view of this, antiseptics show limited bacterial tolerance and have a broad-spectrum antimicrobial activity. However, the application of antiseptics to bone is insufficiently studied in literature. Therefore, we investigated the biocompatibility of the antiseptic polyhexanide with bone related cells and asked whether supplementation to bone cement is appropriate in the management of total arthroplasty infections. METHODS: We performed an in vitro study with immortalised human foetal osteoblast cells (hFOB 1.19) and human endothelial cells (EAhy 926). The cultured cells were exposed to media containing various concentrations of gentamicin (12.5-800 microg/ml) and polyhexanide (0.0006-0.01%) for six hours. We measured the phase-contrast microscopy images, the cell viability, cell number and the alkaline phosphatase activity as a parameter for osteogenic function. RESULTS: The exposure of hFOB and endothelial cells to polyhexanide showed a severe reduction of viability and cell number. Gentamicin did not have negative effects on hFOB and endothelial cell number and viability. The alkaline phosphatase activity of hFOB showed a significant decrease after exposure to polyhexanide and gentamicin. The viability and the cell number of endothelial cells seem more negatively affected by polyhexanide than the parameters of the hFOB-cells. CONCLUSIONS: The exposure of human osteoblasts and endothelial cells to polyhexanide at concentrations with questionable antibacterial activity resulted in severe cell damage whereas exposure to high dosed gentamicin did not. These results raise questions as to the feasibility of using antiseptics in bone cement for the treatment of total arthroplasty infections. Further in vivo studies are necessary to show the in vivo relevance of these in vitro findings.

No increased stem subsidence after arthroplasty in young patients with femoral head osteonecrosis: 41 patients followed for 1-9 years.

BACKGROUND: Poor bone stock in patients with osteonecrosis of the femoral head may be a reason for poor outcome after hip replacement. One way of studying bone quality is to measure implant migration. We thus investigated the clinical and radiographic results of cementless THR in younger patients with femoral head osteonecrosis. PATIENTS AND METHODS: We studied hips in 41 patients (mean age 48 (25-63) years) with a cementless hip arthroplasty after late stage osteonecrosis. Clinical evaluation was by the Harris hip score, the WOMAC score and the SF-36 score. Stem subsidence was measured with the Ein Bild Roentgen Analyse femoral component analysis (EBRA-FCA) at 3, 12, 24, 60, and 72 months after operation. The average duration of follow-up was 7(1-9) years, with less than 2 years for 2 patients. RESULTS: There was no revision of any hip. No radiographic or clinical stem loosening was seen. After 60 months, the cementless stems showed a median subsidence of -0.7 mm (95% CI: -0.9 to -0.2). No femoral osteolysis occurred. Femoral radiolucent lines, all < 1 mm, were seen in 10 hips. At the latest follow-up the Harris hip score was 83 (23-100) points. INTERPRETATION: Our findings for porous-coated stems in patients with femoral osteonecrosis indicate no greater risk of stem subsidence and rate of osteolysis after an average of 7 years follow-up. Thus, we continue to use uncemented stems in younger patients with femoral osteonecrosis. However, continued follow-up will be necessary to evaluate the long-term outcome.

High long-term loosening rate of conical screw cups.

BACKGROUND: Acetabular screw cups seem to give high primary stability. We analyzed the migration and loosening behavior of a first-generation screw cup in a longterm follow-up. PATIENTS AND METHODS: We examined 92 uncemented titanium alloy conical screw cups prospectively. Implant migration was assessed with a digital high-precision method (EBRA) with an accuracy of 1.0 mm. RESULTS: After mean 11 (0.5-18) years, 87 patients were available for examination and 5 patients had died. 32 implants had been revised and 7 cases showed radiographic evidence of loosening. The 10-year survival rate was 71%. Migration of more than 1 mm occurred in 53 hips. Implant survival was strongly associated with an annual migration of greater than 0.2 mm. INTERPRETATION: The long-term behavior of this cup is not satisfactory. In spite of extraordinarily high primary implant stability, secondary osseointegration of this cup often fails. The annual migration rate represents a valid parameter for prediction of implant survival.

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.

BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARgamma, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.

Subsidence of titanium straight stems in combination with highly viscous bone cement.

Varying results and a high rate of subsidence have been reported for the straight femoral stem (M.E. Muller) made of titanium alloy. We examined subsidence in 135 titanium-alloy straight stems implanted with high viscosity cement after 68.8+/-11.5 months using a digital high-precision method (EBRA-FCA). One revised implant showed a subsidence of 14.6 mm and another 2.5 mm over 5 years. A third implant without migration was found to be loose. The 122 implants without loosening showed a mean subsidence of 0.1+/-0.1 mm, and focal osteolysis was seen in two. Altogether, we found subsidence of the titanium stems very small. The small subsidence may be related to the use of high viscosity bone cement.

Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate.

One major problem of current cartilage repair techniques is that three-dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild-type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)-2 or -4 in alginate revealed that the formation of markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP-transfection. Cells were encapsulated in ultrahigh-viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously staining with haematoxylin-eosin or Alcian blue, immunohistochemical analysis, and quantitative RT-PCR confirmed the expression of chondrogenic markers (chondroitin-4- and -6-sulfate as well as type II collagen). Production of chondrogenic markers was particularly high in BMP-4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP-4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild-type cells and to some extent for BMP-2 transfected cells. The osteogenic markers, type I collagen, alkaline phosphatase, and Cbfa1 were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP-4 transfected cells as revealed quantitatively by real time RT-PCR. Thus, the in vitro results suggested that BMP-4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.

Testing of skeletal implant surfaces with human fetal osteoblasts.

The effect of standard orthopaedic implant materials on osteoblast proliferation and differentiation was investigated using a human osteoblast cell culture system. Human fetal osteoblasts 1.19 were cultured on stainless steel, cobalt-chrome-molybdenum, and commercially pure titanium for 12 days. Tissue culture polystyrene was used as a control. Cell proliferation was measured by electronic cell counting and by a colorimetric proliferation assay. To assess the degree of differentiation, levels of alkaline phosphatase activity, collagen Type I, and osteocalcin production were measured. Osteocalcin gene expression was measured by reverse transcriptase-polymerase chain reaction. Electronic cell counting and proliferation assays showed lower cell numbers and delayed proliferation on stainless steel and cobalt-chrome-molybdenum compared with titanium and polystyrene. Alkaline phosphatase and osteocalcin were measured higher on titanium than on stainless steel or cobalt-chrome-molybdenum. Differences in collagen Type I production were not found. Reverse transcriptase-polymerase chain reaction showed the highest osteocalcin gene expression on titanium. The human fetal osteoblast cell line 1.19 provides a rapidly proliferating and differentiating system for testing biomaterials in which differences in osteoblast proliferation and differentiation on orthopaedic implant materials could be revealed, suggesting that the chemistry of biomaterials has a dynamic effect on proliferation and differentiation of human osteoblasts.