From dynamic self-organization to avalanching instabilities in soft-granular threads.

We study the dynamics of threads of monodisperse droplets, including droplet chains and multi-chains, in which the droplets are interconnected by capillary bridges of another immiscible liquid phase. This system represents wet soft-granular matter - a class of granular materials in which the grains are soft and wetted by thin fluid films-with other examples including wet granular hydrogels or foams. In contrast to wet granular matter with rigid grains (e.g., wet sand), studied previously, the deformability of the grains raises the number of available metastable states and facilitates rearrangements which allow for reorganization and self-assembly of the system under external drive, e.g., applied via viscous forces. We use a co-flow configuration to generate a variety of unique low-dimensional regular granular patterns, intermediate between 1D and 2D, ranging from linear chains and chains with periodically occurring folds to multi-chains and segmented structures including chains of finite length. In particular, we observe that the partially folded chains self-organize via limit cycle of displacements and rearrangements occurring at a frequency self-adapted to the rate of build-up of compressive strain in the chain induced by the viscous forces. Upon weakening of the capillary arrest of the droplets, we observe spontaneous fluidization of the quasi-solid structures and avalanches of rearrangements. We identify two types of fluidization-induced instabilities and rationalize them in terms of a competition between advection and propagation. While we use aqueous droplets as the grains we demonstrate that the reported mechanisms of adaptive self-assembly apply to other types of soft granular systems including foams and microgels. We discuss possible application of the reported quasi-1D compartmentalized structures in tissue engineering, bioprinting and materials science.

Distal femoral medial closing wedge osteotomy for degenerative valgus knee : mid-term results in active patients.

Distal femoral medial closing wedge osteotomy (DFMCWO) may be a valuable treatment for arthritic valgus knees in young and active adults, with the possible aim of procrastinating knee replacement. 32 valgus knees (mean age : 41.4±11.2) treated with DFMCWO were retrospectively reviewed. All the knees had a lateral compartment osteoarthritis graded I-II-III according to Kellgren Lawrence classification. 20 knees had osteochondral lesions, treated with microfractures (8) or bone marrow derived cells transplantation (12). Patients were clinically (IKDC, KOOS, NRS, Tegner) and radiologically evaluated. A mean follow-up of 62.12±15.65 was achieved. KOOS score peaked at 24 months, showed a decremental trend, achieved a final results of 79,59±17,14. Similar trend was evident for IKDC. The final NRS score was 2.73±1.82 ; the final Tegner score was 4.81±1.56. Radiographs showed degenerative progression in 5 knees : 2 patients underwent knee replacement at the final follow-up. DFMCWO is an effective treatment to treat osteoarthritic symptomatology, delay degenerative progression and avoid knee replacement in valgus knees at mid-term follow-up.

Failures in bipolar fresh osteochondral allograft for the treatment of end-stage knee osteoarthritis.

PURPOSE: Bipolar fresh osteochondral allografts (BFOA) recently became a fascinating option for articular cartilage replacement, in particular in those young patients non-suitable for traditional replacement because of age. While the use of osteochondral allografts for the treatment of focal osteochondral lesions in the knee is well established, their use in the treatment of end-stage arthritis is far more controversial. The purpose of this paper is to describe our experience in a series of seven patients who underwent a resurfacing of both tibio-femoral and patello-femoral joints by BFOA. METHODS: From 2005 to 2007, seven patients (mean age 35.2 ± 6.3 years) underwent BFOA for end-stage arthritis of the knee. Patients were evaluated clinically, radiographically and by CT scan preoperatively and at established intervals up to the final follow-up. RESULTS: No intra-operative complications occurred. Nevertheless, joint laxity and aseptic effusion, along with a progressive chondrolysis, lead to early BFOA failure in six patients, which were revised by total knee arthroplasty at 19.5 ± 3.9 months follow-up. Only one patient, who received the allograft to convert a knee arthrodesis, gained a satisfactory result at the last follow-up control. CONCLUSIONS: BFOA in the knee joint still remains an inapplicable option in the treatment of post-traumatic end-stage arthritis of the young patient, due to the high rate of failure. Further studies are necessary in order to investigate the causes of failure and improve the applicability of this method. Still, after extensive counselling with the patient, BFOA may represent a salvage procedure aimed to revise scarcely tolerated knee arthrodesis. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Bone marrow concentrated cell transplantation: rationale for its use in the treatment of human osteochondral lesions.

Bone marrow is one of the best characterized stem cell microenvironments that contains Mesenchymal Stem Cells (MSCs), a rare population of non-hematopoietic stromal cells. MSCs have been indicated as a new option for regenerative medicine because of their ability to differentiate into various lineages such as bone, cartilage and adipose tissue. However, isolation procedures are crucial for the functional activity of the transplanted cells. The use of concentrated bone marrow cells (BMCs) enables a cell population surrounded by its microenvironment (niche) to be implanted while avoiding all the complications related to the in vitro culture. The cells of the niche are able to regulate stem cell behavior through direct physical contact and secreting paracrine factors. The aim of this study was to characterize BMCs in vitro to evaluate their ability to differentiate toward mature cells and try to understand whether there are differences in the chondrogenic and osteogenic potential of cells from patients of different ages. Mononuclear Cells (MNCs) isolated by Ficoll were used as control. Both cell populations were grown in monolayers and differentiated with specific factors and analyzed by histological and molecular biology assays to evaluate the expression of some specific extracellular matrix molecules. The present investigations revealed the ability of BMCs to act as isolated cells. They are able to form colonies and differentiate toward chondrogenic and osteogenic lineages, the latter pathway appearing to be influenced by donor age. The results obtained by this study support the use of BMCs in clinical practice for the repair of osteochondral damage, which might be particularly useful for the one-step procedure allowing cells to be directly implanted in operating room.

Treatment of human cartilage defects by means of Nd:YAG Laser Therapy.

Articular cartilage lesions represent a challenging problem for orthopaedic surgeons. The purpose of this study was to evaluate the effect of a new pulsed Nd:YAG High Intensity Laser Therapy on the regeneration of cartilage tissue in patients with traumatic lesions. Clinical, histological and immunohistochemical evaluations were performed. Ten patients affected by chondral lesions scheduled for ACI procedure, were enrolled into the study. During the chondrocyte expansion for ACI procedure, cartilage from five patients was treated by Nd:YAG High Intensity Laser Therapy (HILT group). No laser treatment was performed in the remaining patients, who were used as controls. Cartilage repair was assessed by clinicians using two different scores: Cartilage Repair Assessment (CRA) and Overall Repair Assessment (ORA). Cartilage biopsy specimens were harvested to perform histological and immunohistochemical analyses at T0 (before laser treatment) and T1 (at the end of the treatment). A significant decrease in cartilage depth was noticed in the HILT group at T1. Histological and immunohistochemical evaluations showed some regenerative processes in cartilaginous tissue in terms of high amount of proteoglycans, integration with adjacent articular cartilage and good cellular arrangement in the HILT group. By contrast, a not well organized cartilaginous tissue with various fibrous features in the control group at T0 and T1 was observed. In conclusion, the use of this new pulsed Nd:YAG HILT resulted promising in the treatment of moderate cartilage lesions markedly in the young patients.

Ligament repair: a molecular and immunohistological characterization.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.

Partial ACL tears augmented with distally inserted hamstring tendons and over-the-top fixation: an MRI evaluation.

This study evaluated the mid-term MRI appearance of partial ACL tear augmentation with quadrupled distally inserted hamstrings, while preserving the intact ACL bundle. Twenty-eight patients with ACL partial tear underwent augmentation. After 15-40 months follow-up, patients were evaluated clinically and by MRI. The mean IKDC score at follow-up was 93.8. Twenty-five patients were rated as excellent, three as fair. The mean tibial tunnel section area decreased by 27%. A correlation was noted between the clinical and MRI results: the graft was not visible or continuous with high intensity areas and the mean decrease in the tunnel section area was 3% in the three cases rated as fair. The graft appeared continuous and low intensity and the reduction in tibial tunnel section area was 30% in the cases with excellent clinical results. The residual part of the ACL was still recognizable in 79% of cases. The tibial hamstring attachment appeared normal in 93% of cases. In conclusion, excellent results correlated with a decrease in tunnel size and normal graft appearances on MRI. The poor results showed that the graft was not visible or not continuous, with high intensity areas and intra-ligamentous cystic formation within the tunnel. MRI scanning is useful in evaluating hamstring ACL grafts after reconstruction.

Dissection and characterization of microtubule bundles in the mitotic spindle using femtosecond laser ablation.

The mitotic spindle is a highly organized and dynamic structure required for segregation of the genetic material into two daughter cells. Although most of the individual players involved in building the spindle have been characterized in vitro, a general understanding of how all of the spindle players act together in vivo is still missing. Hence, in recent years, experiments have focused on introducing mechanical perturbations of the spindle on a micron scale, thereby providing insight into its function and organization, as well as into forces acting in the spindle. Among different types of mechanical perturbations, optical ones are more flexible, less invasive, and more precise than other approaches. In this chapter, we describe a detailed protocol for cutting the microtubule bundles in human cells using a near-infrared femtosecond laser. This type of laser microsurgery provides the ability to precisely sever a single microtubule bundle while preserving spindle integrity and dynamics. Furthermore, we describe quantitative measurements obtained from the response of a severed microtubule bundle to laser ablation, which reveal the structure and function of individual parts of the spindle, such as the bridging fiber connecting sister k-fibers. Finally, the method described here can be easily combined with other quantitative techniques to address the complexity of the spindle.

Concentration- and time-dependent effects of gamma-linolenic acid supplementation to tumor cells in culture.

Gamma-linolenic acid (GLA) supplemented to neuroblastoma SK-N-BE, tubal carcinoma TG and colon carcinoma SW-620 cells was incorporated into phospholipids in all the cell lines (although to different extents), in a concentration- and time-dependent manner. All the cell lines were able to metabolize GLA to arachidonic acid, SK-N-BE being the most active. Supplementation with low GLA concentrations for short periods was not sufficient to impair cell proliferation; only higher amounts of GLA had an anti-proliferative effect also in short times. In these conditions, the antiproliferative effect of GLA is probably due to cellular dysfunction caused by fatty acid modifications.

Autologous chondrocyte transplantation in osteochondral lesions of the ankle joint.

The aim of this study was to assess the repair of osteochondral defects of the ankle joint with hyaline cartilage. For this purpose we have been using a technique of autologous chondrocyte transplantation for osteochondral defects of the talus for the last two years. Until the method described in the paper, treatment methods proposed for the repair of cartilaginous defects have not been histologically effective in restoring the hyaline cartilage sheath, and in all cases the neoformation of cartilage was of a fibrocartilaginous nature with varying cellular characteristics. Clinical and histological results obtained using this surgical technique have confirmed its validity. Furthermore, neither subjective nor objective complications have been reported. Less pain and better articular function have also been observed. According to the AOFAS score, an improvement from an average score of 32/100 points pre-op. to 91/100 points at 24 months of follow up was obtained. Laboratory data have confirmed the presence of reconstructed cartilage with chondrocytes and expression of collagen II, characteristic of hyaline cartilage.

Cartilage repair procedures associated with high tibial osteotomy in varus knees: clinical results at 11 years' follow-up.

BACKGROUND AND AIM: In this study, the authors retrospectively compared the clinical outcome of middle-aged active patients affected by a varus knee with medial unicompartmental osteoarthritis: the treatment was isolated high tibial osteotomy (HTO) for one group or HTO associated with a cartilage repair procedure, that was autologous chondrocyte implantation (ACI) for one group (HTO+ACI) and microfracture (MFX) for the other group (HTO+MFX). The purpose of this study was to analyse the clinical outcomes at long-term follow-up. The hypothesis was that a cartilage repair procedure may add some benefit to isolated axial correction. METHODS: 56 patients affected by medial osteoarthcritis in a varus knee were retrospectively investigated: 20 patients were treated by HTO, 18 by HTO+ACI and 18 by HTO+MFX. All patients underwent clinical assessment following HSS and WOMAC rating scores and a radiographic study was performed preoperatively and at follow-up. The statistical analysis confirmed that the three groups of patients were homogeneous regarding clinical, radiographical and anatomopathological patterns; differences have been reported in body mass index (BMI). RESULTS: At final follow-up, improvements in clinical and radiographical results were obtained in all patients. HTO and HTO+ACI series showed significantly higher scores compared to the HTO+MFX series. CONCLUSIONS: At more than 11 years of follow-up, isolated HTO and HTO+ACI treatments showed satisfactory results similar to those reported in the literature with no evidence of superiority of the addition of ACI to isolated HTO. MFX associated with HTO provided the worst results in the series.

Ankle surgery: focus on arthroscopy.

BACKGROUND: The ankle joint can be affected by several diseases, with clinical presentation varying from mild pain or swelling to inability, becoming in some cases a serious problem in daily life activities. Arthroscopy is a widely performed procedure in orthopedic surgery, due to the low invasivity compared to the more traditional open field surgery. The ankle joint presents anatomical specificities, like small space and tangential view that make arthroscopy more difficult. METHODS: From 2000 more than 600 ankle arthroscopies were performed at our institution. The treated pathologies were mostly impingement syndrome and osteochondral lesions, and in lower percentage instabilities and ankle fractures. RESULTS: In the impingement, the AOFAS scores at FU showed an increase compared to scores collected preoperatively, with improvement of symptoms in most of the cases, good or excellent results in 80 % of cases. In ligament injuries, AOFAS score significatively improved at the maximum follow-up. In fractures all patients had an excellent AOFAS score at maximum follow-up, with complete return to their pre-injury activities. In osteochondral injuries, the clinical results showed a progressive improvement over time with  the different performed procedures. Control MRI and bioptic samples showed a good regeneration of the cartilage and bone tissue in the lesion site. CONCLUSIONS: The encouraging obtained clinical results, in line with the literature, show how the arthroscopic technique, after an adequate learning curve, may represent a precious aid for the orthopedic surgeon and for the patient's outcome. LEVEL OF EVIDENCE: Case series, Level IV.

Validity of T2 mapping in characterization of the regeneration tissue by bone marrow derived cell transplantation in osteochondral lesions of the ankle.

OBJECTIVE: Bone marrow derived cell transplantation (BMDCT) has been recently suggested as a possible surgical technique to repair osteochondral lesions. To date, no qualitative MRI studies have evaluated its efficacy. The aim of our study is to investigate the validity of MRI T2-mapping sequence in characterizing the reparative tissue obtained and its ability to correlate with clinical results. METHODS AND MATERIALS: 20 patients with an osteochondral lesion of the talus underwent BMDCT and were evaluated at 2 years follow up using MRI T2-mapping sequence. 20 healthy volunteers were recruited as controls. MRI images were acquired using a protocol suggested by the International Cartilage Repair Society, MOCART scoring system and T2 mapping. Results were then correlated with AOFAS clinical score. RESULTS: AOFAS score increased from 66.8±14.5 pre-operatively to 91.2±8.3 (p<0.0005) at 2 years follow-up. T2-relaxation time value of 35-45 ms was derived from healthy ankles evaluation and assumed as normal hyaline cartilage value and used as a control. Regenerated tissue with a T2-relaxation time value comparable to hyaline cartilage was found in all the cases treated, covering a mean of 78% of the repaired lesion area. A high clinical score was related directly to isointense signal in DPFSE fat sat (p=0.05), and percentage of regenerated hyaline cartilage (p=0.05), inversely to the percentage of regenerated fibrocartilage. Lesion's depth negatively related to the integrity of the repaired tissue's surface (tau=-0.523, p=0.007), and to the percentage of regenerated hyaline cartilage (rho=-0.546, p=0.013). CONCLUSIONS: Because of its ability to detect cartilage's quality and to correlate to the clinical score, MRI T2-mapping sequence integrated with Mocart score represent a valid, non-invasive technique for qualitative cartilage assessment after regenerative surgical procedures.

The detached osteochondral fragment as a source of cells for autologous chondrocyte implantation (ACI) in the ankle joint.

OBJECTIVE: Autologous chondrocyte implantation (ACI) has been successfully used for the treatment of osteochondral lesions of the talus. One of the main problems of this surgical strategy is related to the harvesting of the cartilage slice from a healthy knee. The aim of this study was to examine the capacity of chondrocytes harvested from a detached osteochondral fragment to proliferate and to serve as a source of viable cells for ACI in the repair of ankle cartilage defects. METHODS: Detached osteochondral fragments harvested from the ankle joint of 20 patients with osteochondral lesions of the talus served as the source of human articular cartilage specimens. All of the osteochondral lesions were chronic and of traumatic origin. In all cases, the fragments were utilized to evaluate the viability and proliferation of the cells, the histological appearance of the cartilage tissue and the expression of specific cartilage markers by real-time polymerase chain reaction (PCR). In the 16 patients scheduled for ACI, the expanded chondrocytes were used for chondrocyte implantation. In the other 4 patients, with lesion size <1.5cm(2), microfractures were created during the initial arthroscopic step. As a control group, 7 patients with comparable osteochondral lesions underwent the same surgery, but received chondrocytes harvested from the ipsilateral knee. RESULTS: According to the American Orthopaedic Foot and Ankle Scoring (AOFAS) system, patients in the experimental group had a preoperative score of 54.2+/-16 points and a postoperative one of 89+/-9.6 points after a minimum follow-up time of 12 months (P<0.0005). The control group of patients had a preoperative score of 54.6+/-11.7 points and a postoperative one of 90.2+/-9.7 points at a minimum follow-up time of 12 months (P<0.0005). The clinical results of the two groups did not differ significantly from each other. Chondrocytes isolated from the detached fragments were highly viable, phenotypically stable, proliferated in culture and redifferentiated when grown within the three-dimensional scaffold used for ACI. The morphological and molecular characteristics of the cartilage samples obtained from the detached osteochondral fragments were similar to those of healthy hyaline articular cartilage. CONCLUSIONS: The good results achieved with this strategy indicate that cells derived from the lesioned area may be useful in the treatment of osteochondral defects of the talus.