Impact of the Different Preparation Methods to Obtain Autologous Non-Activated Platelet-Rich Plasma (A-PRP) and Activated Platelet-Rich Plasma (AA-PRP) in Plastic Surgery: Wound Healing and Hair Regrowth Evaluation.

Autologous therapies using platelet-rich plasma (PRP) need meticulous preparation-currently, no standardised preparation technique exists. Processing Quantitative Standards (PQSs) define manufacturing quantitative variables (such as time, volume and pressure). Processing Qualitative Standards (PQLSs) define the quality of the materials and methods of manufacturing. The aim of this review is to use existing PQSs and PQLs to report the in vivo/in vitro results obtained by using different Kits, that utilise different procedures (classified as Closed-Technique and Opened-Technique) to isolate autologous human activated (AA-PRP) or non-activated PRP (A-PRP). PQSs included the volumes of blood collected as well as the reagents used, the time/gravity of centrifugation, and the duration, temperature and tilt level/speed of centrifugation. PQLSs included the use of Calcium Chloride CaCl2, Kit weight, transparency of Kit components, the maintenance of a closed sterile processing environment and the use of a small centrifuge. Eight CE marked devices for PRP extraction were evaluated: Angel®, Biomed®, Cascade® and Selphyl®, Mag-18®, i-Stem®, MyCells® and Regenlab®. Using a Kit with the PQSs and PQLSs described in this study enables the isolation of A-PRP, thereby meeting consensus quality criteria. As our understanding of Critical Quality Attributes (CQAs) of A-PRP continues to evolve, especially with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully help isolate A-PRP of desired CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed.

Recessive mutations in the neuronal isoforms of DST, encoding dystonin, lead to abnormal actin cytoskeleton organization and HSAN type VI.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.

Incidence and Prevention of Cicatricial Scleral Show in the Subciliary Access in Patients With Orbital Fractures.

: Orbital fractures can involve floor, lateral and medial wall. Surgical access depends on fracture's severity, ocular trauma and patient's age. Subciliary, subtarsal, infraorbital or transconjunctival approaches are the main access to the orbit. Surgical interventions in the eyelid may induce scar tissue formation and, consequently, the cicatricial scleral show. The authors present a study with the aim to evaluate the incidence of cicatricial scleral show in patients treated for orbital fractures with or without simultaneous Tarsal Sling Canthopexy in our Plastic Surgery Department. METHODS: The authors evaluated 50 patients divided in 2 groups: Group 1, subciliary approach and reconstruction of orbital floor without simultaneous Canthopexy Tarsal Sling; Group 2: reconstruction of orbital floor through subciliary approach with simultaneous Canthopexy Tarsal Sling. RESULTS: Patients, who underwent Canthopexy Tarsal Sling, did not have any scleral show. Instead patients, who did not undergo this prevention technique, had scleral show even if a minor entity. DISCUSSION: Although there was no muscle or skin removed, in our procedure, but only cutaneous incision, scleral show can appear as a complication. Canthal ligament and tarsus' elasticity influence the incidence of post-surgical scleral show, which is more frequent in elderly patients. Therefore, the authors suggest to prevent it routinely with Tarsal Sling Canthopexy. CONCLUSION: Canthopexy Tarsal Sling is procedure that stretch tarsal structure and it may help to prevent scleral show.

Impact of the Different Preparation Methods to Obtain Human Adipose-Derived Stromal Vascular Fraction Cells (AD-SVFs) and Human Adipose-Derived Mesenchymal Stem Cells (AD-MSCs): Enzymatic Digestion Versus Mechanical Centrifugation.

Autologous therapies using adipose-derived stromal vascular fraction (AD-SVFs) and adult adipose-derived mesenchymal stem cells (AD-MSCs) warrant careful preparation of the harvested adipose tissue. Currently, no standardized technique for this preparation exists. Processing quantitative standards (PQSs) define manufacturing quantitative variables (such as time, volume, and pressure). Processing qualitative standards (PQLSs) define the quality of the materials and methods in manufacturing. The purpose of the review was to use PQSs and PQLSs to report the in vivo and in vitro results obtained by different processing kits that use different procedures (enzymatic vs. non-enzymatic) to isolate human AD-SVFs/AD-MSCs. PQSs included the volume of fat tissue harvested and reagents used, the time/gravity of centrifugation, and the time, temperature, and tilt level/speed of incubation and/or centrifugation. PQLSs included the use of a collagenase, a processing time of 30 min, kit weight, transparency of the kit components, the maintenance of a closed sterile processing environment, and the use of a small centrifuge and incubating rocker. Using a kit with the PQSs and PQLSs described in this study enables the isolation of AD-MSCs that meet the consensus quality criteria. As the discovery of new critical quality attributes (CQAs) of AD-MSCs evolve with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully isolate AD-MSCs of various CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed.

The survey on cellular and tissue-engineered therapies in Europe and neighboring Eurasian countries in 2014 and 2015.

BACKGROUND AIMS: With the support of five established scientific organizations, this report, the seventh of its kind, describes activity in Europe for the years 2014 and 2015 in the area of cellular and tissue-engineered therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis. METHODS: In 2015 [respectively 2014], 205 [276] teams from 32 countries responded to the cellular and tissue-engineered therapy survey; 178 [126] teams reported treating 3686 [2665] patients. RESULTS: Indications were musculoskeletal/rheumatological disorders (32% [33%]), cardiovascular disorders (12% [21%]), hematology/oncology (predominantly prevention or treatment of graft versus host disease and HSC graft enhancement; 20% [20%]), neurological disorders (4% [6%]), gastrointestinal disorders (<1% [1%]) and other indications (31% [20%]). The majority of autologous cells (60% [73%]) were used to treat musculoskeletal/rheumatological (44% [36%]) disorders, whereas allogeneic cells were used mainly for hematology/oncology (61% [68%]). The reported cell types were mesenchymal stromal cells (40% [49%]), chondrocytes (13% [6%]), hematopoietic stem cells (12% [23%]), dermal fibroblasts (8% [3%]), dendritic cells (2% [2%]), keratinocytes (1% [2%]) and others (24% [15%]). Cells were expanded in vitro in 63% [40%] of the treatments, sorted in 16% [6%] of the cases and rarely transduced (<1%). Cells were delivered predominantly as suspension 43% [51%], intravenously or intra-arterially (30% [30%]), or using a membrane/scaffold (25% [19%]). DISCUSSION: The data are compared with those from previous years to identify trends in a still unpredictably evolving field. Perspectives of representatives from plastic surgery practitioners, Iran and ISCT are presented (contributing authors D.A. Barbara, B. Hossein and W.L. Mark, respectively).

Long-term follow-up comparison of two different bi-layer dermal substitutes in tissue regeneration: Clinical outcomes and histological findings.

Double layer dermal substitute (DS) consist of a 3-dimensional collagen structures and a superficial silicon layer that are positioned within the defect provide to promote tissue regeneration in skin wounds. DS often have unique physical characteristics due to differences in manufacturing techniques. The aim of this study is the clinical and histological comparison of Nevelia and Integra double layer DSs in patients with post-traumatic injury wounds. Thirty patients with post-traumatic wounds localised on the inferior limbs were randomised in 2 groups Nevelia or Integra, followed by autologous dermal epidermal graft (DEG). Clinical results were evaluated through the healing time; Manchester Scar Scale (MSS) and Visual Analog Scale (VAS) at 1, 2, and 3 weeks and after 1 and 3 years. Histological and immunohistochemical evaluation were performed at 0, 2, and 3 weeks. The difference in healing time between groups (P = .467, log-rank test), pain and self-estimation was not statistically significant after 35, 42, and 49 days and at 1-year follow up. Histological data showed evident healing of wound after 2 weeks compared with preoperative with both DSs. At 3 weeks reepithelialisation and dermal regeneration were evident with both substitutes; however Nevelia showed early regenerative properties in terms of epidermal proliferation and dermal renewal compared with Integra. Nevelia showed also a more evident angiogenesis vs Integra evaluated as α-SMA immunohistochemistry. Differences in the MSS score were statistically significant at 3 years follow up in favour of Nevelia group (P = .001). At long-term follow up, Nevelia showed a better clinical outcome measured as MSS score vs Integra measured as MSS. Histological and immunohistochemistry data showed that Nevelia allows faster neoangiogenesis and tissue regeneration with neoformed tissue architecture closer to the physiology of the skin.

The use of infrared radiation in the treatment of skin laxity.

BACKGROUND: Cosmetic surgery and medicine are extremely interesting fields for a plastic surgeon. Lasers and lights determine ablation, contraction and regenerating stimulus in skin tissues. The aim of this study is to examine the use of infrared lights in treating facial and body skin laxity. METHODS: Between 2007 and 2011, in the Department of Plastic and Reconstructive Surgery at the Hospital San Camillo-Forlanini, 303 patients were enrolled in the study and underwent laser therapy. The laser operates in wavelength from 1100 to 1800 nm. The treated areas are: face, neck, eyebrows, abdomen, legs and buttocks. RESULTS: We have noticed no systemic complications. A case of a patient with a three days lasting erythema on both lower eyelids caused by laser therapy healed without any pharmacological therapy. Neither hyper- nor hypopigmentation of the skin was found. The satisfaction degree of patients has been: facial, neck and eyebrow lifting "very satisfactory" for 70% of the patients, "satisfactory" for 10%, "unsatisfactory" for 20%; for the other areas it was "very satisfactory" for 40%, "satisfactory" for 20% and "unsatisfactory" for 40%. CONCLUSION: The use of infrared radiation represents a valid alternative to surgical lifting, but cannot replace it. The infrared light technique used has turned out to be useful in contrasting skin laxity of the face and other parts of the body. The absence of scars and surgical risk makes this technique useable for a large number of patients.

Adipose-derived stromal vascular fraction cells and platelet-rich plasma: basic and clinical evaluation for cell-based therapies in patients with scars on the face.

BACKGROUND: Actually, autologous fat grafts have many clinical applications in breast surgery, facial rejuvenation, buttock augmentation, and Romberg syndrome as well as a treatment of liposuction sequelae. OBJECTIVE: The aim of this article was to describe the preparation and isolation procedures for stromal vascular fraction (SVF), the preparation of platelet-rich plasma (PRP), and the clinical application in the treatment of the scar on the face. METHODS: Ten patients with burns sequelae (n = 6) and post-traumatic scars (n = 4) were treated with SVF-enhanced autologous fat grafts obtained by the Celution System. Another 10 patients with burns sequelae (n = 5) and post-traumatic scars (n = 5) were treated with fat grafting based on the Coleman technique mixed with 0.5 mL of PRP.To assess the effects of their treatment, the authors compared their results with those of a control group consisting of 10 patients treated with centrifuged fat. RESULTS: In the patients treated with SVF-enhanced autologous fat grafts, we observed a 63% maintenance of contour restoring after 1 year compared with only 39% of the control group (n = 10) treated with centrifuged fat graft (P < 0.0001). In the patients treated with fat grafting and PRP, we observed a 69% maintenance of contour restoring after 1 year compared with that of the control group (n = 10). CONCLUSIONS: Autologous fat grafting is a good method for the correction of scars on the face instead of the traditional scar surgical excision.

Spontaneous clostridial myonecrosis after pregnancy - emergency treatment to the limb salvage and functional recovery: a case report.

Clostridial myonecrosis (CM) is a rare, life threatening necrotizing infection of a skeletal muscle caused by Clostridium perfringens in the majority of cases. The diagnosis may be difficult because of few diagnostic and cutaneous signs early in its course. Standard therapy involves surgical debridements of a devitalized tissue and high-dose organism-specific antibiotic therapy. The hyperbaric oxygen has also showed its usefulness in the treatment of these infections. Autograft systems as tissue replacement, based on bioengineered materials, have been demonstrated to be safe and effective treatments for chronic wounds and a suitable physiotherapy is recommended for the recovery of functional impairments of upper extremities. We present a rare case of CM of right upper limb treated with a combination of standard treatments and new techniques.

Regenerative surgery of the complications with Morton's neuroma surgery: use of platelet rich plasma and hyaluronic acid.

Morton's neuroma is an entrapment neuropathy of the plantar digital nerve. We treated five patients with wound dehiscence and tendon exposure, after Morton's neuroma surgery excision using a dorsal approach. In this article we describe our technique. From July 2010 to August 2011, at the Department of Plastic and Reconstructive Surgery, University of Rome 'Tor Vergata', five patients (four females and one male), with ages ranging between 35 and 52 years, were treated with a combination of PRP (platelet rich plasma) and HA (hyaluronic acid). Thirty days following surgery, all patients showed a complete healing of the wound. The use of this technique for the treatment of postoperative wound dehiscence and tendon exposure has proven as satisfactory.

Combined use of super-oxidised solution with negative pressure for the treatment of pressure ulcers: case report.

A 61-year-old patient was affected by flaccid paraplegia for 20 years because of post-traumatic medullar injury caused by an accidental fall, with stage IV sacral pressure ulcer for 3 years. The patient later developed stage IV sacral pressure ulcer. After 6 months, a new granulation tissue formation appeared in the wound and a reduction of its diameter was observed (length 20 cm, width 15 cm, depth 5 cm). We therefore treated the wound with PRP (platelet rich plasma) intra-lesion and peri-lesional injections. The wounds were covered with three-dimensional polymerised hyaluronic acid medicated biologic dressing. After the surgery, a moderate reduction in diameter and the depth was observed. Super-oxidised solution (SOS-Dermacyn) was applied to control infection locally together with negative pressure to control the exudate and the local bacteremia, to avoid infectious complications without application of systematic antibiotic therapy.

Skin necrosis of scrotum due to endovascular embolisation: a case report.

The aim of our case report was to analyse the results obtained with the Matriderm® system and autologous skin grafting for the surgical treatment of skin necrosis of scrotum as a result of endovascular embolisation. We recruited one patient with scrotum skin necrosis as a result of endovascular embolisation admitted at the department of Plastic and Reconstructive Surgery, University of Rome 'Tor Vergata'. The patient underwent Matriderm® system and autologous skin grafting for skin necrosis treatment. After a single treatment, reduction of the skin necrosis was obtained, after 30 days from the surgical treatment. Patient experienced a reduction in pain and a complete restoration of the loss in volume and quality of skin was noticed. Matriderm® system and autologous skin grafting is a simple, safe and feasible technique. When comparing this treatment with others, Matriderm® is a simpler, more economic and less time-consuming method, and does not require sophisticated laboratory facilities.

Use of platelet-rich plasma and hyaluronic acid in the loss of substance with bone exposure.

OBJECTIVE: : In lower-extremity surgery, the complex wound with bone exposure remains a challenging problem for the plastic surgeon. The purpose of this study was to describe a new therapeutic approach to stimulate the regeneration of the lower-extremity complex wounds based on a combined treatment composed of platelet-rich plasma (PRP) and hyaluronic acid (HA) dressing. DESIGN: : Wounds with posttraumatic bone exposure have been treated with HA dressing alone or in combination with PRP. PATIENT: : Fifteen patients affected by lower-extremity wound with posttraumatic bone exposure have been treated at the Department of Plastic and Reconstructive Surgery, University of Tor Vergata, Rome, Italy. RESULTS: : After a single treatment, the authors observed that the mean re-epithelialization time was 8.1 weeks in 73.3% patients treated with PRP and HA dressing versus the 30% patients treated with HA dressing only. CONCLUSION: : These data confirm the evidence of using PRP technology in the healing of both soft- and hard-tissue wounds. Moreover, the satisfaction of the patient confirms the quality of this study's results.

The use of MatriDerm® and skin grafting in post-traumatic wounds.

The aim of this study was to prove the effectiveness of MatriDerm(®) combined with skin grafting versus skin grafting alone in post-traumatic wounds treatment. At the Department of Plastic and Reconstructive Surgery of the University of Rome Tor Vergata, we treated 60 patients: 30 patients with dermal substitutes (MatriDerm(®)) combined with autologous skin graft and 30 with skin graft alone. Two weeks after the first treatment, 95% of wounds treated with MatriDerm(®) and skin graft showed a re-epithelisation, whereas it was 75-80% in the control group. We used the Manchester Scar Scale (MSS) and patient's self-estimation scale to assess the outcomes. Mann-Whitney U test was performed for the five items of the MSS and the results were combined to those of patient's self-estimation scale and the re-epithelialisation percentage to test the significance between the two groups. These data confirm the evidence of the clinical use of MatriDerm(®) technology in the healing of soft tissue wounds and prove the effectiveness of combining MatriDerm(®) and skin grafting for the first time. Furthermore, we observed a percentage reduction of wound contraction and in the same time an improvement of elasticity, quality of scars tissue and dermal architecture.

Challenges in the management of chronic wound infections.

OBJECTIVES: Chronic wound infections may delay the healing process and are responsible for a significant burden on healthcare systems. Since inappropriate management may commonly occur in the care of these patients, this review aims to provide a practical guide underlining actions to avoid in the management of chronic wound infections. METHODS: We performed a systematic review of the literature available in PubMed in the last 10 years, identifying studies regarding the management of patients with chronic wound infections. A panel of experts discussed the potential malpractices in this area. A list of 'Don'ts', including the main actions to be avoided, was drawn up using the 'Choosing Wisely' methodology. RESULTS: In this review, we proposed a list of actions to avoid for optimal management of patients with chronic wound infections. Adequate wound bed preparation and wound antisepsis should be combined, as the absence of one of them leads to delayed healing and a higher risk of wound complications. Moreover, avoiding inappropriate use of systemic antibiotics is an important point because of the risk of selection of multidrug-resistant organisms as well as antibiotic-related adverse events. CONCLUSION: A multidisciplinary team of experts in different fields (surgeon, infectious disease expert, microbiologist, pharmacologist, geriatrician) is required for the optimal management of chronic wound infections. Implementation of this approach may be useful to improve the management of patients with chronic wound infections.

Malignant Skin Cancer Excision in Combined Therapy with Electro-Chemotherapy and Dermal Substitute.

Squamous cell carcinoma (SCC) is the second most common malignancy skin cancer. It is characterized by abnormal, accelerated growth of squamous cells (SCs). SCC occurs when DNA damage from exposure to ultraviolet radiation or other damaging agents trigger abnormal changes in the SCs, presenting as painless lesions on areas of high sun exposure, such as the dorsum of the hand and upper extremity. For most skin SCC, the surgical excision alone is standard practice. However, recent efforts in new treatment strategies have involved around adjuvant or concomitant electrochemotherapy (ECT). ECT is a non-thermal tumor ablation modality, safe and effective on any type of solid tumor. An 87-year-old patient affected by hand SCC with invasion of deep structures including tendons was treated with neoadjuvant intra-tumoral ECT sessions followed by a selective surgical removal and reconstruction of the substance loss with collagen dermal template (CDT). Two neoadjuvant intra-tumoral ECT procedures, at distance of 3 months, with the aim to reduce the tumor size before a selective surgery, were performed. This study shows that combined surgical selective excision with ECT and CDT is a valid technique for the extended-deep dorsal hand tumor lesions reconstruction.

Tissue regeneration in loss of substance on the lower limbs through use of platelet-rich plasma, stem cells from adipose tissue, and hyaluronic acid.

BACKGROUND: Platelet-rich plasma (PRP) induces wound regeneration and tissue repair through cell proliferation and differentiation, promoting tissue healing and also acting as an autologous scaffold. With a small quantity of blood, it is possible to obtain the necessary optimal volume of PRP to treat the loss of substance in the lower limb. It has been demonstrated that mesenchymal stem cells are present in the adipose tissue (thus accelerating the effect of the PRP). METHODS: The analysis involved 30 patients with lesions ranging from ulcerative, dystrophic, with substance loss, with differentiating etiopathogenesis all localized on the inferior limb, and to those treated with PRP and autologous fat grafts. The wounds were covered with a 3-dimensional, polymerized hyaluronic acid medicated biologic dressing. The authors' protocol consists of a general checkup; wound examination; instrumental, microbiological, and immunohistochemical diagnostic examinations; and acquisition of photographic images with follow-up at 0, 1, 2, and 3 weeks; 1, 3, and 6 months; and 1 year. RESULTS: The results show an improvement from minor to moderate in 100% of patients after 3 weeks, healing in less than 6 weeks in 47% of patients, and complete wound healing in 57% of patients within 3 months. CONCLUSIONS: The authors' data demonstrate the ability of the combination of PRP and autologous adipose graft to regenerate tissue and epithelialization with wound closure, with a significant healing-time reduction. Furthermore, the minimally invasive technique is well accepted by patients, with a noteworthy improvement of the quality of life along with cost reduction due to the fewer number of medications.

The use of Matriderm and autologous skin grafting in the treatment of diabetic ulcers: a case report.

The aim of our case report was to analyse the results obtained with the Matriderm system and autologous skin grafting for the surgical treatment of diabetic ulcers. We recruited one patient with diabetic ulcers admitted at the Department of Plastic and Reconstructive Surgery, University of Rome 'Tor Vergata'. The patient underwent Matriderm system and autologous skin grafting for diabetic ulcer treatment. After just a single treatment, we obtained reduction in ulcer after 15 days from the surgical treatment. We achieved a reduction in pain and exudate secretion of the ulcer. We noticed an almost complete restoration of the missing volume and good quality of skin. Matriderm system and autologous skin grafting is a simple, safe and feasible technique. This method, when compared with other methods of treatment, is simple, cheap, less time consuming and does not require sophisticated laboratory facilities.

A randomized blinded retrospective study: the combined use of micro-needling technique, low-level laser therapy and autologous non-activated platelet-rich plasma improves hair re-growth in patients with androgenic alopecia.

INTRODUCTION: Mini-invasive therapies based on autologous non-activated Platelet-Rich Plasma (ANA-PRP), Low-Level Laser Therapy (LLL-T), and Micro-Needling Technique (MN-T) used in combining for hair re-growth need to be standardized. OBJECTIVES: The work aims to show in vivo outcomes resulted from retrospective case-series study in which ANA-PRP + MN-T + LLL-T were used in combined in patients affected by Androgenic alopecia. METHODS: 23 patients were treated, of which 13 males were classified in stage I-V by the Norwood-Hamilton scale, and 10 females were classified in stage I-III by the Ludwig scale. Assessment of hair re-growth was evaluated with photography, physician's and patient's global assessment scale, and standardized phototrichograms during a follow-up: T0 - baseline, T1 - 12 weeks, T2 - 23 weeks, T3 - 44 weeks, T4 - 58 weeks. RESULTS: Interesting outcomes represented by a hair density increase of 81 ± 5 hairs/cm2 and 57 ± 7 hairs/cm2 respectively at T1 and T2 compared with baseline (173 ± 5 hairs/cm2 at T1 and 149 ± 9 hairs/cm2 at T2 versus 92 ± 2 hairs/cm2 at baseline) were observed using computerized trichograms. EXPERT OPINION: The main limitation in the autologous regenerative therapies and biotechnologies in hair-regrowth is the extreme variability of PRP products used, in the absence of standardized protocols and widely shared. Appropriate PRP preparations have to be pick after carefully thinking about their bio-molecular specifications and intended indications for use in patients. This approach will aid in matching the optimal PRP product to specific patient factors, leading to improved outcomes and the elucidation of the cost-effectiveness of this treatment. The combined use of biotechnologies as the association of PRP with micro-needling and low-level laser therapy may improve the results in terms of hair count and hair density compared with those obtained by alone PRP. All the procedures must be performed in the full respect of international and local rules. CONCLUSIONS: The effect of the combined use of MN-T, LLL-T, and ANA-PRP has been demonstrated.