Unlocking the depths: multiple factors contribute to risk for hypoxic blackout during deep freediving.

PURPOSE: To examine the effect of freediving depth on risk for hypoxic blackout by recording arterial oxygen saturation (SpO2) and heart rate (HR) during deep and shallow dives in the sea. METHODS: Fourteen competitive freedivers conducted open-water training dives wearing a water-/pressure proof pulse oximeter continuously recording HR and SpO2. Dives were divided into deep (> 35 m) and shallow (10-25 m) post-hoc and data from one deep and one shallow dive from 10 divers were compared. RESULTS: Mean ± SD depth was 53 ± 14 m for deep and 17 ± 4 m for shallow dives. Respective dive durations (120 ± 18 s and 116 ± 43 s) did not differ. Deep dives resulted in lower minimum SpO2 (58 ± 17%) compared with shallow dives (74 ± 17%; P = 0.029). Overall diving HR was 7 bpm higher in deep dives (P = 0.002) although minimum HR was similar in both types of dives (39 bpm). Three divers desaturated early at depth, of which two exhibited severe hypoxia (SpO2 ≤ 65%) upon resurfacing. Additionally, four divers developed severe hypoxia after dives. CONCLUSIONS: Despite similar dive durations, oxygen desaturation was greater during deep dives, confirming increased risk of hypoxic blackout with increased depth. In addition to the rapid drop in alveolar pressure and oxygen uptake during ascent, several other risk factors associated with deep freediving were identified, including higher swimming effort and oxygen consumption, a compromised diving response, an autonomic conflict possibly causing arrhythmias, and compromised oxygen uptake at depth by lung compression possibly leading to atelectasis or pulmonary edema in some individuals. Individuals with elevated risk could likely be identified using wearable technology.

Effects of hyperventilation on oxygenation, apnea breaking points, diving response, and spleen contraction during serial static apneas.

PURPOSE: Hyperventilation is considered a major risk factor for hypoxic blackout during breath-hold diving, as it delays the apnea breaking point. However, little is known about how it affects oxygenation, the diving response, and spleen contraction during serial breath-holding. METHODS: 18 volunteers with little or no experience in freediving performed two series of 5 apneas with cold facial immersion to maximal duration at 2-min intervals. In one series, apnea was preceded by normal breathing and in the other by 15 s of hyperventilation. End-tidal oxygen and end-tidal carbon dioxide were measured before and after every apnea, and peripheral oxygen saturation, heart rate, breathing movements, and skin blood flow were measured continuously. Spleen dimensions were measured every 15 s. RESULTS: Apnea duration was longer after hyperventilation (133 vs 111 s). Hyperventilation reduced pre-apnea end-tidal CO2 (17.4 vs 29.0 mmHg) and post-apnea end-tidal CO2 (38.5 vs 40.3 mmHg), and delayed onset of involuntary breathing movements (112 vs 89 s). End-tidal O2 after apnea was lower in the hyperventilation trial (83.4 vs 89.4 mmHg) and so was the peripheral oxygen saturation nadir after apnea (90.6 vs 93.6%). During hyperventilation, the nadir peripheral oxygen saturation was lower in the last apnea than in the first (94.0% vs 86.7%). There were no differences in diving response or spleen volume reduction between conditions or across series. CONCLUSIONS: Serial apneas  revealed a previously undescribed aspect of hyperventilation; a progressively increased desaturation across the series, not observed after normal breathing and could heighten the risk of a blackout.

Underwater pulse oximetry reveals increased rate of arterial oxygen desaturation across repeated freedives to 11 metres of freshwater.

INTRODUCTION: Recreational freedivers typically perform repeated dives to moderate depths with short recovery intervals. According to freediving standards, these recovery intervals should be twice the dive duration; however, this has yet to be supported by scientific evidence. METHODS: Six recreational freedivers performed three freedives to 11 metres of freshwater (mfw), separated by 2 min 30 s recovery intervals, while an underwater pulse oximeter measured peripheral oxygen saturation (SpO2) and heart rate (HR). RESULTS: Median dive durations were 54.0 s, 103.0 s and 75.5 s (all dives median 81.5 s). Median baseline HR was 76.0 beats per minute (bpm), which decreased during dives to 48.0 bpm in dive one, 40.5 bpm in dive two and 48.5 bpm in dive three (all P < 0.05 from baseline). Median pre-dive baseline SpO2 was 99.5%. SpO2 remained similar to baseline for the first half of the dives, after which the rate of desaturation increased during the second half of the dives with each subsequent dive. Lowest median SpO2 after dive one was 97.0%, after dive two 83.5% (P < 0.05 from baseline) and after dive three 82.5% (P < 0.01 from baseline). SpO2 had returned to baseline within 20 s after all dives. CONCLUSIONS: We speculate that the enhanced rate of arterial oxygen desaturation across the serial dives may be attributed to a remaining 'oxygen debt', leading to progressively increased oxygen extraction by desaturated muscles. Despite being twice the dive duration, the recovery period may be too short to allow full recovery and to sustain prolonged serial diving, thus does not guarantee safe diving.

Case Studies in Physiology: Is blackout in breath-hold diving related to cardiac arrhythmias?

Syncope or "blackout" (BO) in breath-hold diving (freediving) is generally considered to be caused by hypoxia. However, it has been suggested that cardiac arrhythmias affecting the pumping effectivity could contribute to BO. BO is fairly common in competitive freediving, where athletes aim for maximal performance. We recorded heart rate (HR) during a static apnea (STA) competition, to reveal if arrhythmias occur. Four male freedivers with STA personal best (PB) of 349 ± 43 s, volunteered during national championships, where they performed STA floating face down in a shallow indoor pool. A non-coded Polar T31 chest strap recorded R-R intervals and a water- and pressure-proof pulse oximeter arterial oxygen saturation. Three divers produced STA near their PB without problems, whereas one diver ended with BO at 5 min 17s, which was 12 s beyond his PB. He was immediately brought up by safety divers and resumed breathing within 10 s. All divers attained similar lowest diving HR (47 ± 4 beats/min), but HR recordings displayed a different pattern for the diver ending with BO. After a short tachycardia, the three successful divers developed bradycardia, which became more pronounced during the second half of the apnea. The fourth diver developed pronounced bradycardia earlier, and at 2.5 min into the apnea, HR started alternating between approximately 50 and 140 beats/min, until the diver lost consciousness. At resumed breathing, HR returned to baseline. Nadir oxygen saturation was similar for all divers. We speculate that arrhythmia could have contributed to BO, by lowering stroke volume leading to a systolic blood pressure drop, affecting brain perfusion.NEW & NOTEWORTHY Heart rate during prolonged breath-holding until the point of loss of consciousness has not previously been published. The recordings show that blackout was preceded by a period of persistent alterations in R-R intervals, whereby an ectopic beat followed every normal heartbeat. Explanations for this deviating heart rate pattern could be either premature atrial contractions or premature ventricular contractions following every atrial beat, i.e., bigeminy, which could have compromised cardiac pumping function and caused/contributed to blackout.

The Development and Acceptability of a Wilderness Programme to Support the Health and Well-Being of Adolescent and Young Adult Cancer Survivors: The WAYA Programme.

Detailed descriptions of theory, structure, and activities with causal links to specified outcomes of wilderness programs are lacking. Addressing this gap, the present qualitative study gives a thorough description of the development of the Wilderness programme for Adolescent and Young Adult (AYA) cancer survivors (WAYA). WAYA is adapted to the individual needs of AYA cancer survivors. It was conceived around Næss's ecosophy and the Positive Health Model, and refined based on findings from a scoping review and patient/public involvement. Programme aims were to increase physical activity, self-confidence, personal growth, joy, safety within nature, meaningful relationships, and self-efficacy. The programme was an eight-day expedition followed three months later by a four-day base-camp. Activities included hiking, backpacking, kayaking, rock climbing, mindfulness and bushcrafting. Evaluation of the programme through focus group and individual interviews with 15 facilitators and 17 participants demonstrated that a diverse group of participants, challenging activities, and mindfulness-based practices were found to positively influence group bonding and the learning process. Furthermore, including an expedition and base-camp component was found to be beneficial in supporting the development of participants' own personal outdoor practices. In conclusion, this study demonstrated that the WAYA programme is safe and well accepted by AYA cancer survivors.

Protocol of a mixed-method randomised controlled pilot study evaluating a wilderness programme for adolescent and young adult cancer survivors: the WAYA study.

INTRODUCTION: The majority of childhood, adolescent and young adult (AYA) cancer survivors suffer from long-term and late effects such as fatigue, psychological distress or comorbid diseases. Effective health promotion strategies are needed to support the health of this vulnerable group. This protocol provides a methodological description of a study that aims to examine the feasibility and safety of performing a randomised clinical trial (RCT) on a wilderness programme that is developed to support the health of AYA cancer survivors. METHODS AND ANALYSIS: The pilot RCT study has a mixed-method design, including quantitative and qualitative evaluations. Participants are AYAs, aged 16-39 years, that have been diagnosed with cancer during childhood, adolescence or young adulthood. A total of 40 participants will be randomly assigned to a wilderness programme (n=20) or a holiday programme (n=20). Both arms include participation in an 8-day summer programme, followed by a 4-day programme 3 months later. Primary outcomes are feasibility and safety parameters such as time to recruitment, willingness to be randomised, programme adherence and adverse effects. Secondary outcomes include self-reported health such as self-esteem, quality of life, self-efficacy and lived experiences. Descriptive statistics will be used to analyse outcomes and explore indications of differences between the programmes. Interviews are analysed by directed content analysis and hermeneutic phenomenology. A convergent parallel mixed-method analysis design will be applied to integrate quantitative and qualitative data. Results of this feasibility study will inform the preparation for a larger RCT with AYA cancer survivors. ETHICS AND DISSEMINATION: The study protocol is approved by the Swedish Ethical Review Authority (reference: 2020-00239). This study will be performed between January 2021 and December 2023. Results will be published in international peer-reviewed journals, presented at conferences and disseminated to participants, cancer societies, healthcare professionals and outdoor instructors. TRIAL REGISTRATION NUMBER: NCT04761042.

Effects of dynamic apnea training on diving bradycardia and short distance swimming performance.

BACKGROUND: Apnea training enhances bradycardia and improves competitive apnea performance and has been proposed as a training method for other sports, such as swimming. We evaluated the effects of apneic underwater swimming, i.e., dynamic apnea (DYN), in 9 competitive swimmers (TR) who completed ten DYN sessions over 2 weeks. METHODS: TR performed pre- and post-training tests including a static apnea test with continuous heart rate (HR) and peripheral oxygen saturation measurements, all-out 50m and 100m freestyle tests and an all-out DYN test. Control groups were competitive swimmers (SC; N.=10) that trained swimming without DYN, and a non-swimmer group (AC; N.=10) performing only static apnea tests. RESULTS: Post-training, TR mean±SD time for 50 m freestyle improved from 25.51±2.01 s to 24.64±2.02 s (P<0.01) and for 100m from 55.5±4.2 s to 54.1±4.4 s (P<0.05). SC also improved their 100m time from 56.7±3.3 s to 56.0±3.1 s (P<0.01; P=0.07 between groups). Only TR performed DYN tests; DYN distance increased from 62.1±11.5 m to 70.9±18.9 m (P<0.05) while DYN speed decreased from 0.74±0.14m/s to 0.64±0.18 m/s (P<0.01). Static apnea duration did not change in any of the groups, but HR-reduction was enhanced post-training only in TR (24.8±14.8% to 31.1±10.9%, P<0.01; P<0.001 between groups). CONCLUSIONS: We conclude that 2 weeks of DYN training enhanced DYN performance, which may be caused by the enhanced apnea-induced diving bradycardia. Further research is required to determine whether DYN training enhances short distance freestyle swimming performance.

Corrigendum: Using Underwater Pulse Oximetry in Freediving to Extreme Depths to Study Risk of Hypoxic Blackout and Diving Response Phases.

[This corrects the article DOI: 10.3389/fphys.2021.651128.].

When the human brain goes diving: using near-infrared spectroscopy to measure cerebral and systemic cardiovascular responses to deep, breath-hold diving in elite freedivers.

Continuous measurements of haemodynamic and oxygenation changes in free living animals remain elusive. However, developments in biomedical technologies may help to fill this knowledge gap. One such technology is continuous-wave near-infrared spectroscopy (CW-NIRS)-a wearable and non-invasive optical technology. Here, we develop a marinized CW-NIRS system and deploy it on elite competition freedivers to test its capacity to function during deep freediving to 107 m depth. We use the oxyhaemoglobin and deoxyhaemoglobin concentration changes measured with CW-NIRS to monitor cerebral haemodynamic changes and oxygenation, arterial saturation and heart rate. Furthermore, using concentration changes in oxyhaemoglobin engendered by cardiac pulsation, we demonstrate the ability to conduct additional feature exploration of cardiac-dependent haemodynamic changes. Freedivers showed cerebral haemodynamic changes characteristic of apnoeic diving, while some divers also showed considerable elevations in venous blood volumes close to the end of diving. Some freedivers also showed pronounced arterial deoxygenation, the most extreme of which resulted in an arterial saturation of 25%. Freedivers also displayed heart rate changes that were comparable to diving mammals both in magnitude and patterns of change. Finally, changes in cardiac waveform associated with heart rates less than 40 bpm were associated with changes indicative of a reduction in vascular compliance. The success here of CW-NIRS to non-invasively measure a suite of physiological phenomenon in a deep-diving mammal highlights its efficacy as a future physiological monitoring tool for human freedivers as well as free living animals. This article is part of the theme issue 'Measuring physiology in free-living animals (Part II)'.

First Evaluation of a Newly Constructed Underwater Pulse Oximeter for Use in Breath-Holding Activities.

Studying risk factors in freediving, such as hypoxic blackout, requires development of new methods to enable remote underwater monitoring of physiological variables. We aimed to construct and evaluate a new water- and pressure proof pulse oximeter for use in freediving research. The study consisted of three parts: (I) A submersible pulse oximeter (SUB) was developed on a ruggedized platform for recording of physiological parameters in challenging environments. Two MAX30102 sensors were used to record plethysmograms, and included red and infra-red emitters, diode drivers, photodiode, photodiode amplifier, analog to digital converter, and controller. (II) We equipped 20 volunteers with two transmission pulse oximeters (TPULS) and SUB to the fingers. Arterial oxygen saturation (SpO2) and heart rate (HR) were recorded, while breathing room air (21% O2) and subsequently a hypoxic gas (10.7% O2) at rest in dry conditions. Bland-Altman analysis was used to evaluate bias and precision of SUB relative to SpO2 values from TPULS. (III) Six freedivers were monitored with one TPULS and SUB placed at the forehead, during a maximal effort immersed static apnea. For dry baseline measurements (n = 20), SpO2 bias ranged between -0.8 and -0.6%, precision between 1.0 and 1.5%; HR bias ranged between 1.1 and 1.0 bpm, precision between 1.4 and 1.9 bpm. For the hypoxic episode, SpO2 bias ranged between -2.5 and -3.6%, precision between 3.6 and 3.7%; HR bias ranged between 1.4 and 1.9 bpm, precision between 2.0 and 2.1 bpm. Freedivers (n = 6) performed an apnea of 184 ± 53 s. Desaturation- and resaturation response time of SpO2 was approximately 15 and 12 s shorter in SUB compared to TPULS, respectively. Lowest SpO2 values were 76 ± 10% for TPULS and 74 ± 13% for SUB. HR traces for both pulse oximeters showed similar patterns. For static apneas, dropout rate was larger for SUB (18%) than for TPULS (<1%). SUB produced similar SpO2 and HR values as TPULS, both during normoxic and hypoxic breathing (n = 20), and submersed static apneas (n = 6). SUB responds more quickly to changes in oxygen saturation when sensors were placed at the forehead. Further development of SUB is needed to limit signal loss, and its function should be tested at greater depth and lower saturation.

Using Underwater Pulse Oximetry in Freediving to Extreme Depths to Study Risk of Hypoxic Blackout and Diving Response Phases.

Deep freediving exposes humans to hypoxia and dramatic changes in pressure. The effect of depth on gas exchange may enhance risk of hypoxic blackout (BO) during the last part of the ascent. Our aim was to investigate arterial oxygen saturation (SpO2) and heart rate (HR) in shallow and deep freedives, central variables, which have rarely been studied underwater in deep freediving. Four male elite competitive freedivers volunteered to wear a newly developed underwater pulse oximeter for continuous monitoring of SpO2 and HR during self-initiated training in the sea. Two probes were placed on the temples, connected to a recording unit on the back of the freediver. Divers performed one "shallow" and one "deep" constant weight dive with fins. Plethysmograms were recorded at 30 Hz, and SpO2 and HR were extracted. Mean ± SD depth of shallow dives was 19 ± 3 m, and 73 ± 12 m for deep dives. Duration was 82 ± 36 s in shallow and 150 ± 27 s in deep dives. All divers desaturated more during deeper dives (nadir 55 ± 10%) compared to shallow dives (nadir 80 ± 22%) with a lowest SpO2 of 44% in one deep dive. HR showed a "diving response," with similar lowest HR of 42 bpm in shallow and deep dives; the lowest value (28 bpm) was observed in one shallow dive. HR increased before dives, followed by a decline, and upon resurfacing a peak after which HR normalized. During deep dives, HR was influenced by the level of exertion across different diving phases; after an initial drop, a second HR decline occurred during the passive "free fall" phase. The underwater pulse oximeter allowed successful SpO2 and HR monitoring in freedives to 82 m depth - deeper than ever recorded before. Divers' enhanced desaturation during deep dives was likely related to increased exertion and extended duration, but the rapid extreme desaturation to below 50% near surfacing could result from the diminishing pressure, in line with the hypothesis that risk of hypoxic BO may increase during ascent. Recordings also indicated that the diving response is not powerful enough to fully override the exercise-induced tachycardia during active swimming. Pulse oximetry monitoring of essential variables underwater may be an important step to increase freediving safety.

Spleen Size and Function in Sherpa Living High, Sherpa Living Low and Nepalese Lowlanders.

High-altitude (HA) natives have evolved some beneficial responses leading to superior work capacity at HA compared to native lowlanders. Our aim was to study two responses potentially protective against hypoxia: the spleen contraction elevating hemoglobin concentration (Hb) and the cardiovascular diving response in Sherpa highlanders, compared to lowlanders. Male participants were recruited from three groups: (1) 21 Sherpa living at HA (SH); (2) seven Sherpa living at low altitude (SL); and (3) ten native Nepalese lowlanders (NL). They performed three apneas spaced by a two-min rest at low altitude (1370 m). Their peripheral oxygen saturation (SpO2), heart rate (HR), and spleen volume were measured across the apnea protocol. Spleen volume at rest was 198 ± 56 mL in SH and 159 ± 35 mL in SL (p = 0.047). The spleen was larger in Sherpa groups compared to the 129 ± 22 mL in NL (p < 0.001 compared to SH; p = 0.046 compared to SL). Spleen contraction occurred in all groups during apnea, but it was greater in Sherpa groups compared to NL (p < 0.001). HR was lower in Sherpa groups compared to NL both during rest (SL: p < 0.001; SH: p = 0.003) and during maximal apneas (SL: p < 0.001; SH: p = 0.06). The apnea-induced HR reduction was 8 ± 8% in SH, 10 ± 4% in SL (NS), and 18 ± 6% in NL (SH: p = 0.005; SL: p = 0.021 compared to NL). Resting SpO2 was similar in all groups. The progressively decreasing baseline spleen size across SH, SL, and NL suggests a role of the spleen at HA and further that both genetic predisposition and environmental exposure determine human spleen size. The similar HR responses of SH and SL suggest that a genetic component is involved in determining the cardiovascular diving response.

Spleen Volume and Contraction During Apnea in Mt. Everest Climbers and Everest Base Camp Trekkers.

The human spleen can contract and transiently boost the blood with stored erythrocytes. We measured spleen volume and contraction during apneas in two groups, each containing 12 Caucasian participants (each 3 women): one group planning to summit Mt. Everest (8848 m; "Climbers") and another trekking to Everest Base Camp (5300 m; "Trekkers"). Tests were done in Kathmandu (1370 m) 1-3 days after arrival, before the Climb/Trek. Age, height, weight, vital capacity, resting heart rate, and arterial oxygen saturation were similar between groups (not significant). After 15 minutes of sitting rest, all participants performed a 1-minute apnea and, after 2 minutes of rest, 1 maximal duration apnea was performed. Six of the climbers did a third apnea and hemoglobin concentration (Hb) was measured. Three axial spleen diameters were measured by ultrasonic imaging before and after the apneas for spleen volume calculation. Mean (standard deviation) baseline spleen volume was larger in Climbers [367 (181) mL] than in Trekkers [228 (70) mL; p = 0.022]. Spleen contraction occurred during apneas in both groups, with about twice the magnitude in Climbers. Three apneas in six of the Climbers resulted in a spleen volume reduction from 348 (145) to 202 (91) mL (p = 0.005) and an Hb elevation from 147.9 (13.1) to 153.3 (11.3) g/L (p = 0.024). Maximal apneic duration was longer in Climbers [88 (23) seconds vs. 67 (18) seconds in Trekkers; p = 0.023]. We concluded that a large spleen characterizes Climbers, suggesting that spleen function may be important for high-altitude climbing performance.

The Magnitude of Diving Bradycardia During Apnea at Low-Altitude Reveals Tolerance to High Altitude Hypoxia.

Acute mountain sickness (AMS) is a potentially life-threatening illness that may develop during exposure to hypoxia at high altitude (HA). Susceptibility to AMS is highly individual, and the ability to predict it is limited. Apneic diving also induces hypoxia, and we aimed to investigate whether protective physiological responses, i.e., the cardiovascular diving response and spleen contraction, induced during apnea at low-altitude could predict individual susceptibility to AMS. Eighteen participants (eight females) performed three static apneas in air, the first at a fixed limit of 60 s (A1) and two of maximal duration (A2-A3), spaced by 2 min, while SaO2, heart rate (HR) and spleen volume were measured continuously. Tests were conducted in Kathmandu (1470 m) before a 14 day trek to mount Everest Base Camp (5360 m). During the trek, participants reported AMS symptoms daily using the Lake Louise Questionnaire (LLQ). The apnea-induced HR-reduction (diving bradycardia) was negatively correlated with the accumulated LLQ score in A1 (r s = -0.628, p = 0.005) and A3 (r s = -0.488, p = 0.040) and positively correlated with SaO2 at 4410 m (A1: r = 0.655, p = 0.003; A2: r = 0.471, p = 0.049; A3: r = 0.635, p = 0.005). Baseline spleen volume correlated negatively with LLQ score (r s = -0.479, p = 0.044), but no correlation was found between apnea-induced spleen volume reduction with LLQ score (r s = 0.350, p = 0.155). The association between the diving bradycardia and spleen size with AMS symptoms suggests links between physiological responses to HA and apnea. Measuring individual responses to apnea at sea-level could provide means to predict AMS susceptibility prior to ascent.

Similar hyaline-like cartilage repair of osteochondral defects in rabbits using isotropic and anisotropic collagen scaffolds.

Lesions in knee joint articular cartilage (AC) have limited repair capacity. Many clinically available treatments induce a fibrous-like cartilage repair instead of hyaline cartilage. To induce hyaline cartilage repair, we hypothesized that type I collagen scaffolds with fibers aligned perpendicular to the AC surface would result in qualitatively better tissue repair due to a guided cellular influx from the subchondral bone. By specific freezing protocols, type I collagen scaffolds with isotropic and anisotropic fiber architectures were produced. Rabbits were operated on bilaterally and two full thickness defects were created in each knee joint. The defects were filled with (1) an isotropic scaffold, (2) an anisotropic scaffold with pores parallel to the cartilage surface, and (3) an anisotropic scaffold with pores perpendicular to the cartilage surface. Empty defects served as controls. After 4 (n=13) and 12 (n=13) weeks, regeneration was scored qualitatively and quantitatively using histological analysis and a modified O'Driscoll score. After 4 weeks, all defects were completely filled with partially differentiated hyaline cartilage tissue. No differences in O'Driscoll scores were measured between empty defects and scaffold types. After 12 weeks, all treatments led to hyaline cartilage repair visualized by increased glycosaminoglycan staining. Total scores were significantly increased for parallel anisotropic and empty defects over time (p<0.05). The results indicate that collagen scaffolds allow the formation of hyaline-like cartilage repair. Fiber architecture had no effect on cartilage repair.

Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures.

Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three healthy adult donors. Human meniscal fibrochondrocytes (MFCs) were isolated from resected tissue after a partial meniscectomy on a young patient. Passage-4 MFCs were cultured in monolayer for 24 h, and 3 and 7 days. Six different culture media were used containing different amounts of either PRP or PPP and compared to a medium containing 10% FBS. dsDNA was quantified, and gene expression levels of collagen types I and II and aggrecan were measured at different time points with quantitative polymerase chain reaction in the cultured MFCs. After 7 days, the dsDNA quantity was significantly higher in MFCs cultured in 10% and 20% PRP compared to the other PRP and PPP conditions, but equal to 10% FBS. Collagen type I expression was lower in MFCs cultured with medium containing 5% PRP, 10% and 20% PPP compared to FBS. When medium with 10% PRP or 20% PRP was used, expressions were not significantly different from medium containing 10% FBS. Collagen type II expression was absent in all medium conditions. Aggrecan expression did not show differences between the different media used. However, after 7 days a higher aggrecan expression was measured in most culture conditions, except for 5% PRP, which was similar compared to FBS. Statistical significance was found between donors at various time points in DNA quantification and gene expression, but the same donors were not statistically different in all conditions. At 7 days cell cultured with 10% PRP and 20% PRP showed a higher density, with large areas of clusters, compared to other conditions. In an MFC culture medium, FBS can be replaced by 10% PRP or 20% PRP without altering proliferation and gene expression of human MFCs.

Characterization of polyurethane scaffold surface functionalization with diamines and heparin.

Polyurethane scaffolds (PUs) have a good biocompatibility but lack cell recognition sites. In this study, we functionalized the surface of a PU, P(D/L)LA and PCL (50:50) containing urethane segments, with heparin. The first step in this functionalization, aminolysis, lead to free amine groups on the surface of the PU. Free amine content was determined to be 6.4 nmol/mL/mg scaffold, a significant increase of 230%. Subsequently, heparin was crosslinked. Immunohistochemistry demonstrated the presence of heparin homogeneous throughout the 3D porous scaffold. Young's modulus decreased significantly till 50% of the native stiffness after aminolysis and did not change after heparin crosslinking. Contact angle on PU films significantly decreased from 82.7° to 64.3° after heparin crosslinking, indicating a more hydrophilic surface. This functionalization beholds great potential for tissue engineering purposes. When used in a load-bearing environment, caution is necessary due to reduction in mechanical stiffness.

Proliferation of meniscal fibrochondrocytes cultured on a new polyurethane scaffold is stimulated by TGF-ß.

The aim of this study was to investigate if newly developed polyurethane (PU) scaffolds are suitable as scaffold for cell-seeded meniscus tissue engineered constructs. Scaffolds were seeded with goat meniscal fibrochondrocytes and cultured to assess changes in biological and mechanical properties. Furthermore, the effect of TGF-ß on these properties was investigated. PU scaffolds were made from poly d/l lactide and caprolactone as soft segments and 1,4-butanediisocyanate for the urethane hard segments. The porosity of the scaffolds was 95%. Isolated goat meniscal fibrochondrocytes were seeded on the scaffolds and cultured with or without the addition of 10 ng/mL TGF-ß in standard culture medium. After 2, 4, and 6 weeks of culture, scaffolds were analyzed for cell proliferation, matrix synthesis, and mechanical properties. Scanning electron microscopy and histology showed that the scaffolds had an interconnected isotropic pore structure. Without the addition of TGF-ß, cells did not proliferate during the culture period and isolated meniscus fibrochondrocytes were more frequently located in the peripheral parts of the scaffold. Fibrochondrocytes supplemented with TGF-ß were distributed throughout the construct. Clustered cells were surrounded by matrix which stained slightly positive for glycosaminoglycans (GAGs). Also, collagen production was increased significantly after 4 and 6 weeks of culture compared to cultures without TGF-ß and also more GAG staining was found after 4 and 6 weeks in the sections of the TGF-ß stimulated cultures. Despite the increase in matrix production, the compressive stiffness of the constructs was not increased during the culture period. Meniscal fibrochondrocytes were able to adhere to the PU scaffold. However, the scaffold itself does not stimulate proliferation and matrix production. The addition of TGF-ß resulted in a strong induction of both proliferation and extracellular matrix production.

Effect of load on the repair of osteochondral defects using a porous polymer scaffold.

The aim of the present study was to evaluate if a porous polymer scaffold, currently used for partial meniscal replacement in clinical practice, could initiate regeneration and repair of osteochondral defects, and if regeneration and repair were related to mechanical stimulation. Two equally sized osteochondral defects were created bilaterally in each trochlear groove of 16 adult female New Zealand White rabbits. The defects were filled with polycaprolactone-polyurethane scaffolds of either 3 or 4 mm in height. Regeneration and repair of the defects were evaluated after 8 (n = 8) and 14 weeks (n = 8). After 8 weeks of implantation, both the 3- and 4-mm scaffolds were flush with the native cartilage. The amount of cartilaginous tissue was similar in both scaffold types. Pores located in the more central zones of the scaffolds contained less cartilaginous tissue when compared with pores located in the more superficial zones. After 14 weeks, significantly more cartilaginous tissue was present in 4 mm scaffolds when compared with the 3-mm scaffolds (p = 0.03). In the 4-mm scaffolds, progression of cartilaginous tissue from the surface of the scaffold toward the center was observed over time, whereas in the 3-mm scaffold, the percentage of cartilaginous tissue in the central zones was not different from the situation after 8 weeks. Osteochondral defects might be treated using porous polymer scaffolds currently used for partial meniscus replacement, although several limitations need yet to be overcome. The results suggest that mechanical forces may not have to be applied over long periods of time to accelerate tissue formation and increase cartilage repair longevity.