Human platelet lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review.

BACKGROUND: Foetal bovine serum (FBS), is the most commonly used culture medium additive for in vitro cultures, despite its undefined composition, its potential immunogenicity and possible prion/zoonotic transmission. For these reasons, significant efforts have been targeted at finding a substitute, such as serum free-media or human platelet-lysates (hPL). Our aim is to critically appraise the state-of-art for hPL in the published literature, comparing its impact with FBS. MATERIALS AND METHODS: In June 2019 a systematic search of the entire Web of Science, Medline and PubMed database was performed with the following search terms: (mesenchymal stem cells) AND (fetal bovine serum OR fetal bovine calf) AND (human platelet lysate). Excluded from this search were review articles that were published before 2005, manuscripts in which mesenchymal stem cells (MSCs) were not from human sources, and when the FBS controls were missing. RESULTS: Based on our search algorithm, 56 papers were selected. A review of these papers indicated that hMSCs cultured with hPL showed a spindle-shaped elongated morphology, had higher proliferation indexes, similar cluster of differentiation (CD) markers and no significant variation in differentiation lineage (osteocyte, adipocyte, and chondrocyte) compared to those cultured with FBS. Main sources of primary hMSCs were either fat tissue or bone marrow; in a few studies cells isolated from alternative sources showed no relevant difference in their response. CONCLUSION: Despite the difference in medium choice and a lack of standardization of hPL manufacturing, the majority of publications support that hPL was at least as effective as FBS in promoting adhesion, survival and proliferation of hMSCs. We conclude that hPL should be considered a viable alternative to FBS in hMSCs culture-especially with a view for their clinical use.

Anatomically accurate 3D modelling and printing in a case of obstetric brachial plexus injury.

Obstetric brachial plexus injury is reported in 0.42 per 1000 births in UK and Ireland and are associated with a reduction in quality of life for the patient and their carers. In this report we describe the first use of a patient specific, anatomically accurate 3D model as a communication tool in the treatment of a complex case of posterior shoulder subluxation secondary to glenohumeral deformity resulting from obstetric brachial plexus injury. The use of 3D models for surgical planning is associated with decreased operating time and reduction of intra-operative blood loss, whilst their use in patient education increases patient understanding. In this case all surgeons surveyed agreed that it was useful and will use 3D modelling to improve consent processes and to conceptualise novel techniques for complex cases in future. This highly reproducible, low cost technique may be adapted to a variety of upper limb reconstructive surgeries, and as the resolution of image acquisition and additive manufacturing capabilities increase so too do the potential applications of this precise 3D printed surgical adjunct.

Temporary catheter first perfusion during hand replantation with prolonged warm ischaemia.

INTRODUCTION: Since the first successful arm replantation reported by Malt and McKhann in 1962, developments and refinements to upper extremity replantation techniques have led to higher success rates with better functional outcomes. One of the most important determinants of a successful macroreplantation is the ischaemic time of the amputated part, as irreversible muscle necrosis begins after 6 hours of warm ischaemia. With major trauma and plastic surgery units usually covering a wide geographical area, it is often difficult to ensure patient injury to revascularization time is less than 6 hours. In 1981, Nunley et al described the temporary catheter perfusion technique in upper limb replantation surgery to reduce ischaemia time without any significant complications. When used in appropriate cases this technique can reduce complication rates in upper limb replantation surgeries. MATERIAL AND METHODS: Temporary catheter first perfusion was used in a hand replantation after 6 hours of warm ischaemia, with preservation of the intrinsic muscles, as evidenced by return of function. The technique used is described, along with relevant literature. RESULTS: Temporary catheter perfusion allowed early reperfusion of the amputated hand, improving the chance of intrinsic muscle preservation despite delayed presentation. It allowed better wound evaluation and debridement, and facilitated better bone stabilisation prior to vascular repair. CONCLUSION: Temporary catheter perfusion is well described in proximal upper limb replantation procedures. This case shows that it is also a useful adjunct for hand replantation, particularly when the patient presents with a critical duration of warm ischaemia.

Shoulder function following autologous latissimus dorsi breast reconstruction. A prospective three year observational study comparing quilting and non-quilting donor site techniques.

Latissimus dorsi harvest and axillary surgery can affect shoulder function. The effect of autologous latissimus dorsi flap (ALD) breast reconstruction and donor site quilting have been inadequately studied. A cohort of ALD flap breast reconstruction patients were assessed pre-operatively and at eight post-operative time-points (up to 3 years after reconstruction) using the self-administered Disabilities of the Arm, Shoulder and Hand (DASH) outcome measure, for which validated normative data is available. Patients with incidental shoulder conditions and bilateral reconstructions were excluded. This was a prospective, observational study with blinded data interpretation: 58 patients, 22 of whom had donor site quilting, were assessed. Groups were compatible demographically, in breast care management and in pre-operative DASH score (quilted 6.5, non-quilted 6.4; P=0.98). Scores were significantly increased at initial post-operative clinic review (mean 49, SD19; P<0.001), 6 week (29, SD20; P<0.001), and 3 month (19, SD19; P<0.01), thereafter remaining at a plateau value of approximately 15 (P>0.05). Seroma incidence was reduced in the quilted group (5% vs 70%). A strong, significant correlation was found between 3 month DASH score and long term function (r=0.66, P<0.0003); patients with DASH >20 fare significantly worse in the longterm (mean 20 point increase, SD5.0, P<0.001). Higher post-operative DASH scores correlated significantly with pre-operative DASH (r=0.58) and BMI (r=0.36). Adjuvant therapy had no effect on shoulder function. Axillary dissection had a weak correlation with a higher DASH score, but only at the 3-month post-operative time-point (r=0.32, P=0.03). ALD flap breast reconstruction generally results in a functionally insignificant increase (6.5 points) in longterm DASH score, although a small subset of patients do develop longterm impairment, and quilting does not appear to inhibit shoulder function.

Neck management in medullary thyroid carcinoma.

AIMS: The aims of this study were to retrospectively evaluate incidence and patterns of lymph node metastases, surgical treatment and prognostic factors of medullary thyroid carcinoma. METHODS: Out of a group of 70 MTC patients data of 67 patients were collected. Sixty-two of these patients underwent surgery. Apart from thyroidectomy, 16 patients underwent a bilateral neck dissection, 21 a unilateral neck dissection and 29 a paratracheal dissection or node-picking operation. Thirty-six patients were irradiated, of which 31 postoperatively and five with palliative intent. RESULTS: Lymph node metastases were found in 91% of the ipsilateral neck dissection specimens, 91% of the paratracheal dissections and 63% of the contralateral dissections. Of the 12 elective neck dissections, 5 were tumor positive. Level VI was positive in 91% of the cases where a dissection was done, whereas preoperatively only 16% were scored tumor positive. During follow-up 22 of the 67 patients developed one or more locoregional recurrences (in total 28 recurrences). The most important factors that were correlated with a worse prognosis of survival were late stage of disease (stage III and IV) (p=0.0014), high number of positive lymph nodes (p=0.0023) and incomplete surgical resection (p=0.0002). CONCLUSIONS: The high rate of locoregional recurrences in this study are a strong argument for a more aggressive approach to the primary and neck. A routine central and ipsilateral selective neck dissection of levels II-V should be considered in all MTC patients based on the high incidence of metastases and the relative low morbidity of a unilateral neck dissection. Patients referred after thyroidectomy alone with elevated (stimulated) calcitonin levels should be re-operated, performing an elective or therapeutic central and unilateral neck dissection.

Analysis of the dose-response of N-acetylcysteine in the prevention of sensory neuronal loss after peripheral nerve injury.

BACKGROUND: N-Acetylcysteine (NAC) is a safe pharmaceutical agent known to protect cells from oxidative damage. Following peripheral nerve transection, NAC has been found to eliminate sensory neuronal loss. This study examines the dose-response relationship of NAC in preventing neuronal death. METHODS AND FINDINGS: The rat sciatic nerve transection model was used, and stereological quantification of sensory neuron survival carried out at two weeks post-axotomy. NAC was administered systemically as an intraperitoneal injection to five groups of rats at a range of doses (1-300 mg/kg/day). Significant neuronal loss was observed in the 1 mg/kg/day dosage group (18.5% loss, p = 0.067 vs. sham treatment). A degree of neuroprotection occurred with 10 mg/kg/day (9.1% loss, p < 0.005 vs. control), whilst there was no significant loss with either 150 or 300 mg/kg/day. CONCLUSIONS: The prevention of sensory neuronal loss with NAC is dose dependent and effective over a wide therapeutic range. This analysis confirms the efficacy of systemic administration and provides a dose framework with which NAC has clinical potential to improve outcome after peripheral nerve trauma.

The thin circumflex scapular artery perforator flap.

The development of microsurgery has most recently been focused upon the evolution of perforator flaps, with the aim of minimising donor site morbidity, and avoiding the transfer of functionally unnecessary tissues. The vascular basis of perforator flaps also facilitates radical primary thinning prior to flap transfer, when appropriate. Based upon initial clinical observations, cadaveric, and radiological studies, we describe a new, thin, perforator flap based upon the circumflex scapular artery (CSA). A perforator vessel was found to arise within 1.5cm of the CSA bifurcation (arising from the main trunk, or the descending branch). The perforator arborises into the sub-dermal vascular plexus of the dorsal scapular skin, permitting the elevation and primary thinning of a skin flap. This thin flap has been employed in a series of five clinical cases to reconstruct defects of the axilla (two cases of hidradenitis suppurativa; pedicled transfers), and upper limb (one sarcoma, one brachial to radial artery flowthrough revascularisation plus antecubital fossa reconstruction, and one hand reconstruction with a chimeric flap incorporating vascularised bone, fascia, and thin skin flaps; free tissue transfers). No intramuscular perforator dissection is required; pedicle length is 8-10cm and vessel diameter 2-4mm. There was no significant peri-operative complication or flap failure, all donor sites were closed primarily, patient satisfaction was high, and initial reconstructive aims were achieved in all cases. Surgical technique, and the vascular basis of the flap are described. The thin circumflex scapular artery perforator flap requires no intramuscular dissection yet provides high quality skin (whose characteristics can be varied by orientation of the skin paddle), and multiple chimeric options. The donor site is relatively hair-free, has favourable cosmesis and no known functional morbidity. This flap represents a promising addition to the existing range of perforator flaps.

Tertiary resurfacing after one of the first free flaps in Europe, a reflection on 30 years of microsurgical progress.

Free flaps have been used for over 30 years. During this period, improved anatomical understanding has increased donor options and available pedicle lengths, permitting safer, single-stage reconstructions with simpler anastomoses. Refinements, such as perforator flaps in particular, have greatly improved donor morbidity, recipient site cosmesis, and the ability to replace 'like with like' while retaining options for innervation. This case highlights the evolution from one of Europe's first free tissue transfers, effectively a perforator flap, through the advent of free muscle flaps to the current generation of contourable perforator flaps. Free flap transfer has become increasingly sophisticated, safer, and more predictable, yet the potential quality of reconstructive outcome has changed little.

Evaluation outcome of replanted digits using the DASH score: review of 38 patients.

The ultimate usefulness of replanted fingers is related to the adequacy of nerve, tendon and bone repair. Thirty-eight patients with successful replantation of the thumb, a single finger or after multiple digital amputations were followed up clinically. The subjective disability after finger replantation was evaluated by means of the DASH score, and the presence of cold intolerance was assessed. The subjective outcome of the achieved replantation reached an overall DASH score of 12.3. Patients after thumb replantation reached a score of 10.0, after a single finger replantation DASH 11.2 and after multiple finger amputations and replantation of at least one finger, DASH 16.1. Cold intolerance was subjectively found in 86.7% of all hands with replanted fingers without a correlation to the patients DASH scores. The patient's evaluation of their limb function after replanted digits by means of the DASH score, when combined with an objective external assessment, represents a valuable comparative tool.

Motorneuron protection by N-acetyl-cysteine after ventral root avulsion and ventral rhizotomy.

Motor recovery after proximal nerve injury remains extremely poor, despite advances in surgical care. Several neurobiological hurdles are implicated, the most fundamental being extensive cell death within the motorneuron pool. N-acetyl-cysteine almost completely protects sensory neurons after peripheral axotomy, hence its efficacy in protecting motorneurons after ventral root avulsion/rhizotomy was investigated. In adult rats, the motorneurons supplying medial gastrocnemius were unilaterally pre-labelled with retrograde tracer (true-blue/fluoro-gold), prior to L5 and 6 ventral root avulsion, or rhizotomy. Groups received either intraperitoneal N-acetyl-cysteine (ip, 150 or 750 mg/kg/day), immediate or delayed intrathecal N-acetyl-cysteine treatment (it, 2.4 mg/day), or saline; untreated animals served as controls. Either 4 (avulsion model) or 8 (rhizotomy model) weeks later, the pre-labelled motorneurons' mean soma area and survival were quantified. Untreated controls possessed markedly fewer motorneurons than normal due to cell death (avulsion 53% death; rhizotomy 26% death, P<0.01 vs. normal). Motorneurons were significantly protected by N-acetyl-cysteine after avulsion (ip 150 mg/kg/day 40% death; it 30% death, P<0.01 vs. no treatment), but particularly after rhizotomy (ip 150 mg/kg/day 17% death; ip 750 mg/kg/day 7% death; it 5% death, P<0.05 vs. no treatment). Delaying intrathecal treatment for 1 week after avulsion did not impair neuroprotection, but a 2-week delay was deleterious (42% death, P<0.05 vs. 1-week delay, 32% death). Treatment prevented the decrease in soma area usually found after both types of injury. N-acetyl-cysteine has considerable clinical potential for adjuvant treatment of major proximal nerve injuries, including brachial plexus injury, in order that motorneurons may survive until surgical repair facilitates regeneration.

Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.

Breast-milk sodium as a predictor of breastfeeding patterns.

This study partially replicates and extends a study reporting that elevated breast-milk sodium BM [Na+] during early lactogenesis was predictive of poor breastfeeding outcomes. The present study used 6-day postpartum breast milk. Consistent with the findings of the earlier study, 80% of those with a BM [Na+] of 16 mmol/L or lower at day 6 sustained a high level of breastfeeding at week 4, compared to only 50% of those with an elevated BM [Na+] (chi 2 = 4.05, df = 1, p = .04). This difference was even greater in a subgroup of mothers predicted to be at high risk for insufficient milk supply on the basis of support density and self-perception variables. Of the latter group, 75% with low BM [Na+] sustained a high level of breastfeeding at 4 weeks postpartum, compared to only 22% with an elevated BM [Na+] (chi 2 = .65, df = 1, p = .01). In contrast, among the low-risk mothers BM [Na+] levels were not associated with any difference in breast-milk sustainment (89% and 82% sustainment for low- and high-sodium groups, respectively). Thus a normal drop in BM [Na+] is predictive of higher sustainment of breastfeeding. However, the predictive validity of this marker appears to be enhanced by combining it with the psychosocial variables of support density and self-perception of breastfeeding by the mother.

Enhancement of hepatitis C virus NS3 proteinase activity by association with NS4A-specific synthetic peptides: identification of sequence and critical residues of NS4A for the cofactor activity.

The NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor for cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region of the viral polyprotein. To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage sites, synthetic peptides spanning various parts of NS4A were synthesized and tested in a cell-free trans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates. The NS3 proteinase domain was expressed in Escherichia coli, purified, denatured, and refolded to an enzymatically active form. We found that a 12-amino-acid peptide containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis. The peptide enhanced the cleavage at the NS5A/5B site and was necessary for NS3-mediated cleavage at NS4A/4B and NS4B/5A. Sequential amino acid substitution within the designated peptide identified residues I29 and I25 as critical for potential cofactor activity. We provide evidence that the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex. These data suggest that the central 12-amino-acid peptide (aa 22-33) of NS4A is primarily important for the cofactor activity through complex formation with NS3, and the interaction may represent a new target for antiviral drug development.

Breathing measurement reduces false-negative classification of tachypneic preextubation trial failures.

OBJECTIVES: There is increased awareness of imposed work of breathing contributing to apparent ventilatory dependency. This study evaluates the impact of tachypnea as an indicator of ventilatory failure during a room air-5 cm H2O continuous positive airway pressure, spontaneous breathing, preextubation trial when associated with increased imposed work of breathing. DESIGN: Prospective, descriptive, 1-yr data collection. SETTING: University hospital trauma intensive care unit (ICU). PATIENTS: Mechanically ventilated trauma ICU patients surviving to discharge. INTERVENTION: Patients were weaned to minimal mechanical ventilator support and underwent a 20-min room air-continuous positive airway pressure preextubation trial (FIO2 = 0.21, continuous positive airway pressure = 5 cm H2O [0.5 kPa]). When passed (PaO2 >/= 55 torr [>/= 7.3 kPa], PaCO2 /= 7.35, respiratory rate 1.1 joule/L, imposed work of breathing was measured, and if residual "physiologic" work of breathing (patient work of breathing minus imposed work of breathing) was 30 breaths/min. Of these, 97 were successfully extubated despite tachypnea ranging from 32 to 56 breaths/min, when combined with either a patient work of breathing

Cell-free expression of the coxsackievirus 3C protease using the translational initiation signal of an insect virus RNA and its characterization.

We have expressed the 3C protease of coxsackievirus B3 (CVB3) in a cell-free system. This expression system employs the translational initiation signal of an insect virus RNA, black beetle virus (BBV) RNA 1, to direct CVB3-specific protein synthesis. Using this expression system, we demonstrate that a biologically active 3C protease is synthesized which possesses both cis and trans processing capabilities. This in vitro-synthesized 3C protease is analogous to the native 3C, which was obtained from cytoplasmic extracts of CVB3-infected HeLa cells, in all biological parameters that were evaluated. In addition, antibody prepared against the 3C protease purified from extracts of CVB3-infected HeLa cells cross-reacts with the 3C protease produced in this cell-free system. Using the translational initiation signal from BBV RNA 1, we also have expressed the CVB3 capsid precursor and part of the P2 region in vitro, and have shown that the capsid precursor is cleaved between 1C (VP3) and 1D (VP1) by the proteolytic activity of in vitro-synthesized 3C in trans. Evidence also is presented to implicate the 2A protein of CVB3 as having proteolytic function.